The One Health approach emphasizes the interconnectedness of human health, animal health, and the environment, particularly in addressing zoonotic diseases. Leptospirosis, caused by the bacteria of the genus Leptospira, is a significant zoonotic disease with global implications. This study focuses on investigating leptospirosis in lemurs, small mammals, and domestic animals within zoo settings in Madagascar, considering the complexities of disease transmission in such environments. This research aims to assess the prevalence of leptospirosis in both free-ranging and enclosed lemurs, along with introduced mammals like invasive rodents and domestic animals, across rainy and dry seasons. The hypotheses to be tested are: (1) Enclosed lemurs are at a higher risk of leptospirosis due to potential exposure to rats and dogs within zoo premises, and (2) Leptospirosis prevalence is higher during the rainy season compared to the dry season.  The study will be conducted at Ivoloina Zoological Park in Madagascar. Capture and sample collection methods will include netting lemurs for physical exams and blood/urine sampling, trapping domestic animals, and using Sherman traps for rodents. Laboratory analyses will involve serology and PCR techniques for pathogen detection. Statistical analysis will determine prevalence rates and associated risk factors. The findings of this study will highlight the risk of leptospirosis transmission in zoo environments, emphasizing the need for effective disease control measures. Additionally, insights gained from this research will inform future surveillance efforts for other rodent-borne pathogens, contributing to improved zoo management, public health, and wildlife conservation.