As a large and structurally diverse family of small molecules, bile acids play a crucial role in regulating lipid, glucose, and energy metabolism. In the human body, bile acids share a similar chemical structure with many isomers, exhibit little difference in polarity, and possess various physiological activities. The types and contents of bile acids present in different diseases vary significantly. Therefore, comprehensive and accurate detection of the content of various types of bile acids in different biological samples can not only provide new insights into the pathogenesis of diseases but also facilitate the exploration of novel strategies for disease diagnosis, treatment, and prognosis. The detection of disease-induced changes in bile acid profiles has emerged as a prominent research focus in recent years. Concurrently, targeted metabolomics methods utilizing high-performance liquid chromatography-mass spectrometry (HPLC-MS) have progressively established themselves as the predominant technology for the separation and detection of bile acids. Bile acid profiles will increasingly play an important role in diagnosis and guidance in the future as the relationship between disease and changes in bile acid profiles becomes clearer. This highlights the growing diagnostic value of bile acid profiles and their potential to guide clinical decision-making. This review aims to explore the significance of bile acid profiles in clinical diagnosis from four perspectives: the synthesis and metabolism of bile acids, techniques for detecting bile acid profiles, changes in bile acid profiles associated with diseases, and the challenges and future prospects of applying bile acid profiles in clinical settings.