Human mammary cell lines are extensively used for preclinical safety assessment of insulin analogs. However, it is essentially unknown how mitogenic responses can be optimized in mammary cell-based systems. We developed an insulin mitogenicity assay in MCF-7 human mammary adenocarcinoma cells, under low serum (0.1% FCS) and phenol red-free conditions, with 3H thymidine incorporation as endpoint. Based on EC50 values determined from 10-fold dilution series, beta-estradiol was the most potent mitogen, followed by human IGF-1, human AspB10 insulin and native human insulin. AspB10 insulin was significantly more mitogenic than native insulin, validating the ability of the assay to identify hypermitogenic human insulin analogs. With MCF-7 cells on a collagen IV surface, the ranking of mitogens was maintained, but fold mitogenic responses and dynamic range and steepness of dose-response curves were increased. Also, PI3K pathway activation by insulin was enhanced on a collagen IV surface. This study provided the first determination and ranking of the mitogenic potencies of standard reference compounds in an optimized MCF-7 assay. The optimized MCF-7 assay described here is of relevance for in vitro toxicological testing and carcinogenicity safety assessment of new insulin compounds.