Steroid hormone profiling has historically underpinned advances in endocrine investigation and research, crucially dependent on selective and sensitive hormone assays. Mining the "steroidome" by mass spectrometry (MS) provides greater specificity than immunoassays. Building on a 50 year legacy, gas and liquid chromatography-MS continue to evolve (e.g. sequential derivatisation, mobile phase modifiers). Exciting new technology (e.g. imaging, ion mobility, supercritical fluid), sample preparation (microextraction, molecular imprinted polymers) are advancing the field. Automated analysis of wider profiles of steroids is within reach, in smaller and more varied sample types, including molecular mapping of tissue sections. These new analytical dimensions require rigorous validation and advanced statistics. This review highlights that MS continues to open new windows of biochemical understanding in endocrinology.