Chromatographic method has long been recognized as the most widely used separation method in bioanalytical research. However, the relatively low sensitivity of existing chromatographic methods remains a significant challenge, as the requirements for experimental procedures become more demanding. This review discusses the main causes for the low sensitivity of chromatographic methods and aims to introduce different technologies for enhancing their sensitivity in the following aspects: (i) different pretreatment methods for improving clean‐up efficiency and recovery; (ii) derivatization step for altering the chromatographic behavior of analytes and enhancing MS ionization efficiency; (iii) optimal LC–MS conditions and appropriate separation mechanism; and (iv) applications of other chromatographic methods, including miniaturized LC, 2D‐LC, 2D‐GC, and supercritical fluid chromatography. Altogether, this review is devoted to summarizing the recent technologies reported in the literature and providing new strategies for the detection of bioanalytes.