In vitro culture of rodent spermatogonial stem cells (SSCs) has become an important asset in the study of mammalian SSC biology. Supported by added growth factors, SSCs divide in culture and form aggregates of stem/progenitor spermatogonia, termed clusters. Recent studies have shown that serial passaging of clusters results in long-term maintenance and amplification of the SSC pool and that this culture system can also be used for short-term semiquantification of SSC activity. Here, we report the development of an automated assay to assess the activity of rat stem/progenitor spermatogonia in vitro and its application for investigating the cytotoxicity of chemotherapeutic drugs on these cells. Cultures of EGFP-expressing rat spermatogenic cells allowed us to determine the number and two-dimensional surface area of clusters using an automated fluorescence imaging system, thereby providing quantitative data of SSC activity. Using this assay, we examined the germ cell toxicity of three drugs that are routinely used in testicular cancer therapy, namely, bleomycin, cisplatin, and etoposide, alone and in combination. All three drugs showed a significant and dose-dependent reduction of cluster number and surface area, indicating their adverse effects specific to spermatogonia. The inhibitory concentration at which cluster number and surface area are inhibited by 50% (IC₅₀) was the lowest with etoposide and the highest with cisplatin, implying that etoposide was most toxic to spermatogonia in vitro. These results suggest that the SSC culture should provide an effective and efficient system to assess the germ cell toxicity of various drugs and chemical compounds.