Estrogens play an important role in human breast carcinogenesis and are of clinical importance. Currently, the most widely used techniques are automated immunoassays, owing to the fast sample throughput and minimal sample preparation required. However, these techniques suffer from poor accuracy and precision at low concentration levels, and are susceptible to interferences. The objective of this study was to develop a highly sensitive method for the analysis of estrogens in serum, without derivatization, using the QSight® 420 LC/MS/MS system.