After Holmes has carefully examined the three bedrooms at Stoke Moran, he tells Helen that she must sleep in her old bedroom and that he and Watson will spend the night in the bedroom she has recently been occupying, directly next to her stepfather's. She is to place a light in the window of that room as a signal that her stepfather has retired for the night and that she is moving to her former bedroom. Holmes and Watson are watching the house from their rooms at the nearby Crown inn.

About nine o'clock the light among the trees was extinguished, and all was dark in the direction of the Manor House. Two hours passed slowly away, and then, suddenly, just at the stroke of eleven, a single bright light shone out right in front of us.

So Holmes and Watson climb through the bedroom window at eleven o'clock. They sit in the pitch-dark room next to Dr. Roylott's keeping perfectly silent. 

Far away we could hear the deep tones of the parish clock, which boomed out every quarter of an hour. How long they seemed, those quarters! Twelve struck, and one and two and three, and still we sat waiting silently for whatever might befall.

Dr. Roylott apparently killed Helen's sister Julia two years before by sending his snake through the ventilator, where it would crawl down the dummy bell-pull and onto the sleeping girl's bed. According to what Julia had told Helen, she had been hearing a low whistling sound for three nights in a row at around three o'clock in the morning; and on the fourth night she died in her sister's arms in the corridor outside her bedroom. When Helen is talking to Holmes at Baker Street that morning, she says that what alarmed her was hearing the low whistling sound described by her sister. Helen does not state what time it was, but it was not long before daylight. Dr. Roylott used the whistle to call the snake back through the ventilator. He was doing this with Julia at about three o'clock each morning because he did not want her to wake up and find a snake in bed with her. He had trained the snake to respond to the whistle. So while Holmes and Watson are waiting in the dark they hear that same low whistle.

The instant that we heard it, Holmes sprang from the bed, struck a match, and lashed furiously with his cane at the bell-pull.

The angry snake bites Dr. Roylott and kills him. Thus Holmes has protected his client Helen from being murdered and solved the mystery of her sister's death. He explains to Watson:

Of course he must recall the snake before the morning light revealed it to the victim. He had trained it, probably by the use of the milk which we saw, to return to him when summoned. He would put it through this ventilator at the hour that he thought best, with the certainty that it would crawl down the rope and land on the bed. It might or might not bite the occupant, perhaps she might escape every night for a week, but sooner or later she must fall a victim.

Early in the story Holmes mentions the cold weather several times. Roylott's snake comes from the warm climate of India. It seems probable that it would crawl right under the covers with the sleeping girl and curl up beside her warm body. Julia had been sleeping with a poisonous snake for three nights before she was bitten. On the fourth night she apparently had set a candle and a box of matches near her bed so that she could strike a light if she heard the whistle. Helen tells Holmes:

"In her right hand was found the charred stump of a match, and in her left a match-box.”

Julia must have turned over in bed to reach for the matches and rolled right on top of the lethal snake. It would have bitten her through her nightgown, but she might not have been aware of it. She got up to light the candle, freeing the snake to climb back up the bell-pull in response to the whistle.