Tamoxifen (TMX), a selective estrogen receptor modulator, is commonly used in the treatment of hormoneresponsive cancers. However, the effects of TMX in anabolic tissues harboring estrogen receptors, such as skeletal muscle, are poorly understood. We report a tandem mass-tag approach to TMXtreated myogenesis in C 2 C 12 cells, a well-characterized model of in vitro murine skeletal muscle differentiation. A longitudinal analysis of >10,000 proteins identified in untreated C 2 C 12 myogenesis revealed a novel subset of 1,062 myogenically regulated proteins. These proteins clustered into five distinct longitudinal expression trends which significantly overlap those obtained in similar analyses performed in human myocytes. We document a specific functional enrichment for adiponectin-signaling unique to TMX-treated myogenesis, as well as a subset of 198 proteins that are differentially expressed in TMX-treated cells relative to controls at one or more stages of myogenesis, the majority of which were involved in steroid and lipid metabolism. Further analysis highlights metallothionein-1 as a novel target of TMX treatment at each stage of C 2 C 12 myogenesis. Finally, we present a powerful, selfvalidating pipeline for analyzing the total proteomic response to in vitro treatment across every stage of muscle cell development which can be easily adapted to study the effects of other drugs on myogenesis.