This pioneering study is prompted by the imperative to enhance our understanding of a constituent within Ukraine's rabies control strategy, specifically focusing on the vaccination of free-roaming canines against rabies with a local vaccine in certain endemic rabies areas. The cross-sectional study encompassed the capture, sterilization, deworming, and rabies vaccination processes in 160 free-roaming dogs, followed by the collection of blood samples to examine RABV-specific neutralising antibodies in 16 dogs (10% of the vaccinated population), captured from sectors (territories) with a higher density of population and with higher density of previously vaccinated free-roaming dogs. Half of the targeted samples comprised males (n = 8), while the remaining half consisted of females (n = 8). The median of virus-neutralising antibody level was 0.58 IU/mL, with a minimum protective threshold of 0.5 IU/mL. Antibody titers below the 0.5 IU/mL threshold were detected in 25.0% (2/8) of the male group, and in 62.5% (5/8) of the female group. Notably, male dogs exhibited a higher median antibody level of 0.66 IU/mL, compared to females, who presented a median level of 0.26 IU/mL. However, no statistically significant difference was found between the male and female groups (P = 0.36). In general, more than half of the tested population (56.3%) exceeded the 0.5 IU/mL protective threshold 4 months post-vaccination. The inadequate levels of antibodies neutralizing the rabies virus are likely a result of a confluence of factors, including stressors such as nutritional and temperature-related challenges, along with variations in the responses of individual immune systems. Considering the endemic rabies situation and large population of free-roaming dogs in Ukraine, we suggest repeated vaccination for free-roaming dogs against rabies one year after the previous vaccination. In perspective, we suggest conducting large-scale epidemiological studies to assess the impact of animal-related, vaccine-related, and environment-related parameters on the efficacy of rabies vaccines used in Ukraine.