Steroid hormones and their metabolites are currently undergoing clinical trials as potential therapeutics for traumatic brain injury (TBI). To support this work, it is necessary to develop improved procedures for differentiating isobaric species in this compound class. Equilin sulfate (E-S), estrone sulfate (E1-S), 17α-dihydroequilin sulfate (ADHE-S), and 17β-dihydroequilin sulfate (BDHE-S) are primary constituents in hormone replacement therapies, such as Premarin, which are among pharmaceuticals being investigated for TBI treatment. The latter three compounds are isomers and can be difficult to differentiate in trace analytical determinations. In this work, a systematic study of the fragmentation of ADHE-S, BDHE-S, E1-S, and E-S under different stages of higher order tandem mass spectrometry (MS(n)) and variation of collision energy, allowed optimization of conditions for distinguishing the isomeric structures. For epimeric variants (e.g., ADHE-S versus BDHE-S; α- versus β-stereoisomerization in the C-17 position), differentiation was achieved at MS(4) and fragmentation was demonstrated through MS(5). Computational analysis was performed to further explore differences in the fragmentation pathways due to changes in stereochemistry.