•LC/ESI–MS/MS has been widely used for the quantification of endogenous steroids.•However, the ESI efficiency and fragmentation behavior of some steroids are poor.•Chemical derivatization is effective for enhancing detectability of steroids.•This article reviews chemical derivatization for LC/ESI–MS/MS of steroids.•Stable isotope-coded derivatization for the steroid analysis is also described. Sensitive and specific methods for the detection, characterization and quantification of endogenous steroids in body fluids or tissues are necessary for the diagnosis, pathological analysis and treatment of many diseases. Recently, liquid chromatography/electrospray ionization–tandem mass spectrometry (LC/ESI–MS/MS) has been widely used for these purposes due to its specificity and versatility. However, the ESI efficiency and fragmentation behavior of some steroids are poor, which lead to a low sensitivity. Chemical derivatization is one of the most effective methods to improve the detection characteristics of steroids in ESI–MS/MS. Based on this background, this article reviews the recent advances in chemical derivatization for the trace quantification of steroids in biological samples by LC/ESI–MS/MS. The derivatization in ESI–MS/MS is based on tagging a proton-affinitive or permanently charged moiety on the target steroid. Introduction/formation of a fragmentable moiety suitable for the selected reaction monitoring by the derivatization also enhances the sensitivity. The stable isotope-coded derivatization procedures for the steroid analysis are also described.