This study evaluated the seroconversion response to rabies vaccination in dogs in the Valle del Cauca department, Colombia, in 2009. The department has been rabies-free for over 20 years, but sylvatic rabies foci remain a threat. Annual vaccination campaigns are conducted to prevent the spread of the disease. The study aimed to measure the impact of vaccination on neutralizing antibody seroconversion and humoral immune response, and to correlate these responses with various factors related to the vaccination process and logistics. The study included 569 vaccinated dogs from 42 municipalities, with serum samples analyzed using quantitative ELISA. The results showed that 9.1% of the dogs were seronegative, and 25.1% did not exhibit an adequate humoral immune response to vaccination. Antibody concentrations gradually decreased over time, correlating with dog age and vaccine quality but not with gender or breed. The study concluded that to increase the percentage of seropositive dogs with adequate humoral immune response, the following recommendations are suggested: 1. Use viable, non-expired rabies vaccines. 2. Administer two doses of vaccine during the first six months of a dog's life. 3. Administer booster vaccinations at least once a year. 4. Health authorities should regularly inspect practices and processes related to private vaccination institutions. The findings provide valuable insights for local and national health authorities to improve rabies vaccination programs in other Colombian departments and similar regions.This study evaluated the seroconversion response to rabies vaccination in dogs in the Valle del Cauca department, Colombia, in 2009. The department has been rabies-free for over 20 years, but sylvatic rabies foci remain a threat. Annual vaccination campaigns are conducted to prevent the spread of the disease. The study aimed to measure the impact of vaccination on neutralizing antibody seroconversion and humoral immune response, and to correlate these responses with various factors related to the vaccination process and logistics. The study included 569 vaccinated dogs from 42 municipalities, with serum samples analyzed using quantitative ELISA. The results showed that 9.1% of the dogs were seronegative, and 25.1% did not exhibit an adequate humoral immune response to vaccination. Antibody concentrations gradually decreased over time, correlating with dog age and vaccine quality but not with gender or breed. The study concluded that to increase the percentage of seropositive dogs with adequate humoral immune response, the following recommendations are suggested: 1. Use viable, non-expired rabies vaccines. 2. Administer two doses of vaccine during the first six months of a dog's life. 3. Administer booster vaccinations at least once a year. 4. Health authorities should regularly inspect practices and processes related to private vaccination institutions. The findings provide valuable insights for local and national health authorities to improve rabies vaccination programs in other Colombian departments and similar regions.