Syphilis remains a global health challenge requiring accurate Rapid Plasma Reagin (RPR) titer monitoring; however, visual results are susceptible to procedural variations. This study aimed to analyze the impact of incubation temperature (15°C and 25°C) and rotation time (6, 8, and 10 minutes) on RPR titers at the North Sulawesi Regional Health Laboratory (BLKD). All available RPR-reactive serum samples during the study period were included as study samples. RPR testing was performed at incubation temperatures of 15°C and 25°C, with rotation times of 6, 8, and 10 minutes. This study adopt a quasi-experimental design, 30 reactive serum samples were analyzed using non-parametric Wilcoxon Signed-Rank and Friedman tests. Results demonstrated a significant effect of incubation temperature on RPR titers (p = 0.001), where suboptimal temperature (15°C) reduced flocculation intensity. Regarding rotation time, the Friedman test indicated overall differences (p = 0.007). In conclusion, incubation temperature significantly affects titer accuracy, making laboratory temprature control crucial, while variations in rotation time yield significantly different effects in this study.