Purpose The objective of the present study was to evaluate the efficacy of pasteurization, freezing, the addition of formaldehyde and peroxymonosulfate on the inactivation of the bovine leukemia virus (BLV) present in milk. Materials and Methods A sheep bioassay was carried out in 40 sheep, which were intraperitoneally inoculated with leukocytes from milk infected by the BLV previously treated with one of the virus inactivation methods. Five study groups were evaluated: (1) control group: milk without previous treatment, (2) pasteurization group: milk treated by pasteurization, (3) freezing group: milk treated by freezing for 36 hours, (4) formaldehyde group: 0.1% formaldehyde, and (5) peroxymonosulfate group: 0.05% peroxymonosulfate. The inoculated animals were followed for 10 weeks. Results At week 10 post-inoculation, all the animals (8/8) of the control group and the peroxymonosulfate group were seropositive to BLV, while no animals were seropositive (0/8) to BLV in the remaining three groups. Statistically significant differences were found between the pasteurization, freezing and formaldehyde groups with respect to the control (p<0.001) and peroxymonosulfate groups (p<0.001). Conclusion The results indicate that pasteurization, freezing and formaldehyde processes are efficient in inactivating the BLV and can be used in milk to prevent the transmission of the virus.