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Summary: The growing interest in clinical diagnostics has recently focused on metabolic biomarkers. Here, we present a protocol for sample preparation, extraction of cholesterol-related sterols, and quantification of 10 sterols in human blood serum samples using targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). We also describe steps of machine learning techniques to develop novel decision-making systems that offer potential benefits in disease monitoring and surveillance by measuring metabolic pathways.For complete details on the use and execution of this protocol, please refer to Kočar et al.1 and Skubic et al.2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.