A highly sensitive and selective quantitative method to accurately determine testosterone (Te) and 5alpha-dihydrotestosterone (DHT) in human serum is crucial to the success of Te replacement therapy for hypogonadism. To this end we have developed and validated a semi-automated and relatively high-throughput method in a 96-well plate format using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC/MS/MS) for the simultaneous determination of Te and DHT in human serum. Te and DHT along with the internal standards [(2)H(3)]-Te and [(2)H(3)]-DHT were extracted from 300 microL of human serum by liquid-liquid extraction using methyl tertiary-butyl ether (MTBE), followed by derivatization with 2,3-pyridinedicarboxylic anhydride and solid-phase extraction for sample clean up. A novel chemical derivatization approach using 2,3-pyridinedicarboxylic anhydride was employed to achieve the MS sensitivity and selectivity required for DHT. Baseline separation of Te and DHT derivatives from endogenous steroid derivatives was achieved using UPLC technology on a C18 stationary-phase column with 1.7 microm particle size. The validity of using double charcoal-stripped female human serum as surrogate matrix for preparation of calibration standards was demonstrated through standard addition experiments. The method was validated over the concentration ranges of 0.2-40 ng/mL for Te and 0.01-2 ng/mL for DHT. The validation and study sample analysis results show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in 1 day.