Jean-Francois Perreault, 41, and Imran Zafar Syed, 39, were arrested in 2016, minutes before a flight from Glasgow to Toronto was due to take off.
Blood tests were said to have found them above the legal alcohol limit.
But the case collapsed when prison staff destroyed some of the samples.
The case was due to call at Paisley Sheriff Court on Thursday, but prosecutors decided to offer no evidence against the men and the hearing was cancelled.
The Canadian pair were due to fly an Air Transat plane with 250 passengers on board when they were arrested at Glasgow Airport on 18 July 2016.
They were taken to Helen Street Police Station in Glasgow, where they were interviewed and blood samples were taken.
The charge stated that Captain Perreault had 32 milligrams of alcohol in 100ml of blood, while First Officer Syed had 49 milligrams in 100ml of blood.
The blood alcohol limit for flying is set at 20 milligrams.
They were also accused of acting in a threatening manner and fighting at the Hilton Hotel in the city on the same day.
At the police station each blood sample was split in two – one of each for the prosecution and one for each pilot.
The pilots’ samples were placed within their property, as they were in custody, and remained there during their initial court appearance, during which they were remanded in custody.
When the pilots arrived at Low Moss Prison in East Dunbartonshire, prison guard Paul Scoular said he thought the samples would be worthless evidentially and, along with prison nurse Mara Fraser, destroyed them.
Mr Scoular, 46, gave evidence at a hearing in September, which can only be reported now the case has concluded.
He said he was “unsure” what to do as he had “no previous experience of receiving a blood sample in to custody” during his 20-year career working at Scottish jails and he said no official protocol existed.
Mr Scoular said: “My understanding was they should’ve been stored and kept in a cooled environment.
“I asked Mara Fraser whether the samples would still have integrity or whether there may have been a deterioration due to them not being stored in a chilled environment.
“I spoke to Mr Syed and Mr Perreault and raised my concerns. I wasn’t sure if it was worth retaining the sample.”
He did not seek the advice of the police, prosecutors, or managers – and told the pilots he was going to destroy them.
He added: “There was no facility in the prison for storing them. They both gave me a verbal consent to destroy it.”
Prison nurse Mara Fraser, 54, said in evidence: “I informed him [Paul Scoular] I’d have to find out what to do with them.
“I put them in to a locked drawer in the nurses’ office. None of the nurses knew what to do.
“I didn’t know the protocol. I later found out the patients were liberated so I destroyed them [the samples] – I put them in to a sharps bin.”
Lawyers for the pilots argued their right to a fair trial was compromised because there was now no opportunity for them to have the samples independently tested.
A Crown Office spokesman said: “It is the duty of the Crown to keep cases under review.
“After full and careful consideration of the facts and circumstances, including the recent decision of the Court of Appeal, Crown Counsel instructed there should be no further proceedings at this time.
“We are working with Police Scotland to ensure there are proper processes and guidance in place covering the retention and storage of samples when an accused person is remanded in custody.”
Mr Perreault, of Ontario and Mr Syed of Toronto had denied all the charges against them.
Both men were suspended by their employer Air Transat following their arrest.
Glasgow-based TV presenters and interior designers, Colin McAllister and Justin Ryan, were due to be on the flight to Canada.
They explained that passengers had been told the plane could not take off due to “operational reasons”.
Mr Ryan, who along with Mr McAllister presented a TV show in Canada, said it was a “real hassle” that their flight had been delayed “first by a couple of hours, then by 21 hours, and then cancelled”.
The flight eventually lifted off at 10:30 the following day and passengers received compensation from the airline.>