Author summary Visceral leishmaniasis caused by the parasite Leishmania infantum is a neglected tropical disease transmitted from animal hosts to humans by sand fly bites. This potentially fatal disease affects thousands of people annually and threatens millions who live in disease risk areas. Domestic dogs are considered as the main reservoir of this parasite which can also cause a severe chronic canine disease. Allopurinol is the main drug used for long term treatment of this disease but it often does not eliminate infection in dogs. We have recently demonstrated that allopurinol resistant parasites can be isolated from naturally infected dogs that have developed clinical recurrence of disease during allopurinol treatment. In this study we aimed to see if resistance can be induced in susceptible parasite strains isolated from sick dogs by growing them in increasing drug concentrations under laboratory conditions. The changes in allopurinol susceptibility were measured and the impact of drug on parasite growth was monitored over 23 weeks. Induction of resistance was successful producing parasites 20-folds less susceptible to the drug. The pattern of change in drug susceptibility suggests that a genetic change is responsible for the increased resistance which is likely to mimic the formation of resistance in dogs.