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In human body, the endocrine system, nervous system and immune system jointly shoulder the important responsibility of life support. Hormones are the material basis for coordination of endocrine system. Steroid hormones, also known as steride hormone, belong to a large category of hormones. The synthesis process of steroid hormones takes cholesterol as the precursor. Through the participation of various metabolic enzymes, steroid hormones are synthesized in the human adrenal cortex, mitochondria of placental cells, smooth Endoplasmic reticulum and gonads, producing 21C, 19C or 18C-backbones with different chemical structures. According to pharmacological effects, steroid hormones are mainly divided into progestogens, corticosteroids and sex hormones, which play an important role in regulating human growth, development, reproduction and maintaining the stability of human body environment. In clinical practice, the level of steroid hormones is used as diagnostic indicator for adrenal and psychiatric diseases. Therefore, accurate determination of steroid hormone content is very important. Steroid hormones in the human body can be detected using various methods, such as radioimmunoassay, chemiluminescence immunoassay, enzyme-linked immunosorbent assay, gas chromatography-tandem mass spectrometry (GC-MS/MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mass spectrometry has obvious advantages in terms of detection sensitivity and accuracy compared with immunoassay. GC-MS has been the main technique for steroid analysis due to its high specificity, wide analyte coverage, and sufficient sensitivity. However, the samples need to be derivatized, and the experimental processes are tedious. LC-MS/MS has the inherent advantages of high sensitivity and specificity, and has a high-throughput characteristic, thus it is an ideal tool for routine diagnosis. At present, common pretreatment techniques for serum samples include protein precipitation, liquid-liquid extraction, and solid phase extraction. Some new pretreatment methods have emerged in recent years, such as dispersed liquid-liquid extraction, ultrasonic-assisted extraction, and supramolecular extraction. Each pretreatment technique has its own advantages and disadvantages. For example, solid phase extraction (SPE) can achieve good recovery but requires more time and economic factors. In selection of pretreatment methods, the physical and chemical properties of analytes, the characteristics of matrices, the simplicity and durability of method establishment, and time, reliability, and cost required should be comprehensively considered. This article reviewed the application of LC-MS/MS in the determination of endogenous steroid hormones, especially in sample pretreatment, chromatographic and mass spectrometric conditions, so as to provide more accurate basis for clinical diagnosis and treatment based on steroid hormones.