In recent decades the number of men facing reproductive health and fertility problems has increased. Male reproductive dysfunction can be a result of exposure to chemicals during critical periods of testicular development. Multiple studies have shown that testis development is one of the most sensitive developmental processes and often drives risk assessment. The testis, however, is difficult to study and methods for assessing toxicity remain limited. This study addressed gaps in toxicity testing for this sensitive organ by building upon a cell culture method and framework for assessing toxicity outcomes in the developing testis. The present study 1. developed a systems biology platform for integrating normal and adverse responses across testis development in rodents in vivo and in vitro 2. leveraged a previously developed primary culture method to quantify baseline characteristics of the mouse culture in vitro and 3. used the primary mouse culture to evaluate adverse effects of cadmium treatment during a critical window of susceptibility. The systems biology platform was created by carrying out a literature search to create a framework for anchoring proliferation, steroid regulation and spermatogenesis processes throughout testis development. The co-culture for evaluating baseline and toxicant response to cadmium was initiated by isolating testis tissue from immature mice on postnatal day (PND) 9. To evaluate the ability of the in vitro culture to capture dynamic developmental processes, we characterized long-term viability, testosterone production, protein expression and morphology up to 16 days in culture. Western blotting revealed expression of cell type-specific protein markers and microscopy indicated changes in morphology throughout time. Additionally, testosterone detected in the culture indicated a switch in testosterone production from fetal to adult Leydig cells. To evaluate testis response to cadmium, the culture was treated on days in vitro (DIV) 2, 6 and 15 and effects were measured 24 hours later. These time points were chosen based on sensitive developmental processes shown to be happening in vivo in the systems biology framework. Cadmium was introduced to the culture with 2.5, 5 and 10 µM concentrations. We observed a dose dependent disruption in Leydig cell proliferation and testosterone production as well as a decrease in spermatogenesis processes on DIV 7 and 16, 24 hours post cadmium treatment. These quantitative results have been interpreted within our systems biology platform and demonstrate the potential of our model to capture multiple adverse outcomes in proliferation, steroid regulation and spermatogenesis pathways of male reproductive development.