Testosterone, the major androgenic hormone in humans, is commonly measured to aid in the diagnosis of clinical conditions related to its excess or deficiency. In addition, testosterone measurements are used to monitor testosterone replacement-, or antiandrogen therapy. Most commonly, automated direct immunoassays have been used to measure testosterone in human serum. Their advantage compared with other methodologies, lies in high- and rapid sample throughput with minimal human intervention. However, many automated testosterone immunoassays suffer from poor accuracy at the low concentration levels (<50 ng/dL) seen in women and children, or in men undergoing anti-androgen therapy. Our objective was to develop a LC–MS/MS method which measures testosterone in human serum while fulfilling the following criteria: Rapid pre-analytical sample processing with minimal manual sample manipulation; Minimize sample volume requirements; Accurate, precise and unambiguous measurement; Functional sensitivity of 5–10 ng/dL; Sample throughput of at least 30 samples per hour. Our validation criteria for precision, accuracy, and linearity was to have accuracy and linearity within mean limits of ±10%; Intra and inter-assay precision of <15% throughout the reporting range. We also wanted to compare our results to a previously validated LC–MS/MS assay which utilized a manual liquid–liquid extraction and to an automated commercial immunoassay (Bayer ACS:180). We describe here a sensitive and rapid testosterone assay based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) utilizing on-line sample extraction and multiplexing.