Lyme borreliosis, a multisystemic disease caused by spirochetes of the genus Borrelia, is the most common tick-borne disease in the northern hemisphere. Differently from human medicine, several vaccines are available for dogs. To provide the best protection possible, vaccination schemes should be adapted regularly to meet the needs resulting from an increased tick exposure risk due to an inescapable climate change. In this retrospective study, a total of 183 vaccinations were performed with a commercial, multivalent vaccine against Lyme borreliosis, and vaccinated dogs were monitored over an observation period of 13 months. Dogs were either vaccinated on days 0 and 21 and a booster on day 365 (standard vaccination schedule), or with an additional booster vaccination on day 180. Canine serum samples were then tested for their borrelia-specific antibody levels using a two-tiered test system consisting of a kinetic ELISA followed by a line immunoassay. Dogs vaccinated with the standard vaccination schedule displayed decreasing antibody levels between days 120 and 360, which is probably insufficient to prevent an infection with borreliae. In contrast, the additional booster vaccination received on day 180 intercepts this decline in antibody levels between days 225 and 360, providing a sufficient immunity to prevent infection. The results from this retrospective study allow us to recommend a basic vaccination schedule with an additional booster vaccination on day 180 to ensure the best possible protection for dogs against Lyme borreliosis.