<p>Steroids are a class of lipids sharing a cyclopentanoperhydrophenanthrene skeleton, and they play a critical role in the diagnosis and monitoring of numerous endocrine pathologies. However, steroid analysis faces unique challenges, primarily due to their low biological concentrations, poor ionization efficiencies, and high structural similarities—especially among isomers. Herein, we describe a novel analytical strategy that combines Girard’s P (GirP) derivatization of steroids with electron-activated dissociation (EAD) in MS² to enable effective isomer differentiation. Four sets of steroid isomers were investigated, including three regioisomeric pairs (testosterone/dehydroepiandrosterone (DHEA), 11-deoxycortisol/17-deoxycortisol, aldosterone/cortisone) and one epimeric pair (androsterone/epiandrosterone). Under EAD conditions, the constitutional isomer generates unique diagnostic ions, which are used for specific identification and accurate quantification. In contrast, the differentiation of epimeric pairs remains unresolved in our methodology. Notably, these diagnostic ions cannot be produced via low-energy collision-induced dissociation (CID), highlighting the advantage of EAD. Furthermore, we characterized and quantified steroid isomer compositions in mouse serum and various mouse tissues.</p> Graphical Abstract <p></p>