The second test, taken at the request of trainer Bob Baffert, means the horse is a step closer to becoming just the second Derby winner to later be disqualified in the race's 147-year history.
Medina Spirit won the Kentucky Derby on May 1 by a half-length over Mandaloun. Medina Spirit's win marked a record seventh Derby win for Baffert.
However, a post-race drug test showed that the horse tested positive for betamethasone, a banned substance. Following the positive test, Churchill Downs suspended Baffert from racing horses at the track.
Baffert claims that Medina Spirit had been treated with Otomax earlier this year for a case of dermatitis. He said the vet that applied the ointment was unaware that betamethasone was an active ingredient in the medication.
The only horse to be disqualified for medication after winning the Derby is Dancer's Image in 1968, the Associated Press reports.
According to the AP, Medina Spirit is Baffert's fifth horse to fail a drug test in the past year. Several racing commissions, including the Arkansas Racing Commission, have penalized Baffert in recent months following positive drug tests.
Medina Spirit finished third in the Preakness Stakes last month, ending his Triple Crown bid. In mid-May, the New York Racing Association suspended Baffert from entering horses at Belmont Park, meaning the horse could not compete at the upcoming Belmont Stakes.