Bisphenol A (BPA), an endocrine disruptor, was shown to be a testicular toxicant in animal models. Its structural analogs were introduced to the market as BPA alternatives. However, their reproductive toxicological data are limited. Traditional reproductive toxicity testing is expensive and time-consuming with extensive animal use. High-content analysis (HCA) has emerged as a powerful tool for chemical toxicity profiling and screening. This dissertation project aimed to use HCA assays to examine testicular toxicities of BPA and its analogs, bisphenol S (BPS), bisphenol AF (BPAF), and tetrabromobisphenol A (TBBPA) in single testicular cell type cultures and a testicular cell co-culture model.HCA assays were developed and validated in mouse spermatogonial cell line C18-4. HCA revealed that BPAF and TBBPA exhibited higher toxicities than BPA or BPS, including reduction of cell number, alteration in nuclear morphology, perturbation of cell cycle progression and cytoskeleton, and induction of DNA damage responses. A machine learning (ML)-powered HCA pipeline was developed to determine phenotypic changes in a testicular cell co-culture model utilizing mouse spermatogonial, Leydig, and Sertoli cell lines. Exposure to BPA and its analogs resulted in the loss of spatial arrangement of three-dimensional (3D) structure and M phase arrest in the co-cultures. BPAF induced multinucleated cells, which were associated with increased DNA damage responses and impaired actin cytoskeleton. Multiple HCA endpoints measured in the co-cultures also showed BPAF and TBBPA exerted higher toxicities than BPA or BPS. The HCA-based comparative toxicity profiling of BPA and its analogs in mouse spermatogonial, Leydig, and Sertoli cell lines showed cell-specific toxicities of tested chemicals. BPA and BPS exerted higher toxicities in Sertoli and Leydig cells, as compared to spermatogonial cells. BPAF exhibited distinct toxicity profiles among three types of cells at different doses. TBBPA showed low toxicity in Leydig cells. In conclusion, BPA and its analogs affect multiple cellular and molecular events and show differential toxicities in three types of testicular cells and co-cultures, with testicular toxicities being cell-type specific. The newly developed HCA assays, together with the ML algorithm, can be used in identification, screening, and comparison of potential reproductive toxins.