As analytical technologies in proteomics and metabolomics continue to mature, there is an increasing need to apply these to clinically relevant biologic samples. In this study, a liquid chromatography-tandem mass spectrometry method that utilizes selected reaction monitoring was used to measure the absolute quantity of estrogens and estrogen metabolites and testosterone in 8-microm tissue sections obtained from a fresh frozen lymph node tumor infiltrated by metastatic breast carcinoma. Total (conjugated plus unconjugated) and unconjugated levels of these steroid hormones were measured using two cohorts, each containing five adjacent serial sections cut from this tumor. The results were highly reproducible across replicate samples, showing that typical histological tissue sections represent an important sample type for the measurement of these specific metabolites.