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Measuring the concentrations of steroid hormones in plasma is critical for understanding their role in various vital physiological processes. The detection of underivatized steroid hormones in biofluids through mass spectrometry (MS) is typically hindered by low ionization efficiency. We described a novel matrix-assisted laser desorption/ionization-MS (MALDI-MS) approach based on hydroxylamine derivatization (HA-D) to analyze low-concentration steroid hormones in plasma. The ketonic carbonyl group containing steroid hormones could be de