The word "ambition" appears only three times in William Shakespeare's Macbeth. Macbeth himself uses the word only once. The reason that the word appears so few times in the play is that Macbeth's ambition is never questioned.
It's clear to the audience that Macbeth is ambitious from as early in the play as act 1, scene 3, when the three Witches appear to Macbeth and Banquo and give them prophecies. The ones most relevant to Macbeth's ambition are the prophecy made to Macbeth that he "shalt be King hereafter" and the one made to Banquo: "Thou shalt get kings, though thou be none."
Macbeth's first spoken reaction to the prophecies is to the prophecy made to Banquo, which gives the audience the first clue to Macbeth's ambition.
MACBETH. Your children shall be kings.
Macbeth is surprised at the news from Ross and Angus that he's been made Thane of Cawdor, which seems to confirm the truth of the prophecies the Witches made to him. (Duncan actually made Macbeth Thane of Cawdor in act 1, scene 2. Macbeth is just now hearing about it.)
Macbeth isn't surprised by the ambition to be King that this news arouses in him, but he's surprised by the depth of his ambition and the extent to which he's prepared to go to achieve it.
MACBETH. ... why do I yield to that suggestion
Whose horrid image doth unfix my hair
And make my seated heart knock at my ribs,
Against the use of nature? ...
My thought, whose murder yet is but fantastical,
Shakes so my single state of man that function
Is smother'd in surmise.
Macbeth tries to put the murderous thoughts out of his mind—"If chance will have me king, why, chance may crown me / Without my stir"—but those thoughts will return soon enough.
In act 1, scene 4, Duncan names his son, Malcolm, as his heir and his successor to the throne of Scotland, and this announcement almost immediately reignites Macbeth's ambition. He speaks of Duncan making Malcom his heir:
MACBETH. That is a step
On which I must fall down, or else o'erleap,
For in my way it lies. Stars, hide your fires;
Let not light see my black and deep desires.
Nothing that Macbeth does in pursuit of his ambition from this point forward in the play will come as a surprise to the audience.
What does come as a surprise is Lady Macbeth's ambition. In act 1, scene 5, Lady Macbeth enters the scene reading a letter from Macbeth telling her about the witches and their prophecies. Lady Macbeth's response is immediate.
LADY MACBETH. Glamis thou art, and Cawdor, and shalt be
What thou art promised.
Lady Macbeth is well-aware of Macbeth's ambition. Her only concern is that he won't act on it.
LADY MACBETH. Thou wouldst be great;
Art not without ambition, but without
The illness should attend it.
A messenger enters to tell Lady Macbeth, "The King comes here tonight," and Lady Macbeth reveals the true depth of her own ambition. She calls on evil spirits to "unsex me here / And fill me, from the crown to the toe, top-full / Of direst cruelty!" She wants the spirits to remove her humanity from her so that she can do whatever needs to be done to make Macbeth King, and she already has a pretty good idea of what needs to be done.
Lady Macbeth needn't have been concerned about Macbeth's resolve in pursuing his ambition. There's a moment, however, in act 1, scene 7, when Macbeth seems indecisive about going forward with his and Lady Macbeth's plan to kill Duncan, and he questions his ambition
MACBETH. I have no spur
To prick the sides of my intent, but only
Vaulting ambition, which o'erleaps itself
And falls on the other.
Macbeth lists a number of reasons why he shouldn't kill Duncan, but these reasons aren't about killing Duncan, per se, but about killing Duncan at this particular time. Macbeth's reasons not to kill Duncan are based on possible repercussions and retributions against him, political expediency, and what he anticipates will be a outcry from the people in reaction to the killing of a beloved king.
At no time, however, does Macbeth question the morality of killing Duncan. This issue never arises.
Lady Macbeth quickly turns Macbeth from doubt to action, saying, "But screw your courage to the sticking-place, / And we'll not fail." From this point on in the play, Macbeth's ambition is never questioned.
Only one use of the word "ambition" occurs after Macbeth murders Duncan, and the reference isn't to Macbeth, but to Duncan's two sons, Malcolm and Donalbain, who are believed to have killed their own father.
MACDUFF. Malcolm and Donalbain, the King's two sons,
Are stol'n away and fled, which puts upon them
Suspicion of the deed.
ROSS. ’Gainst nature still!
Thriftless ambition, that wilt ravin up
Thine own life's means!
Macduff doesn't believe for a minute that anyone but Macbeth killed Duncan, and possibly neither does Ross. Ross says that it's pointless for Malcolm and Donalbain to have killed Duncan, since they gained nothing by killing him ("Thriftless ambition"), and they had to flee Scotland for fear of the loss of their own lives.
Banquo, too, knew from the moment that Macbeth said "Your children shall be kings" (1.3.89), if not from before, that Macbeth was ambitious to be King. Banquo never questioned Macbeth's ambition, and he had no doubt where Macbeth's ambition would lead.
BANQUO. Thou hast it now: King, Cawdor, Glamis, all,
As the weird women promised, and I fear
Thou play'dst most foully for't.
With that last line, Banquo prophesies Macbeth's own demise.