Electrospray ionization coupled with collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS) is a commonly used technique to analyze the chemical composition of steroids. However, steroids are structurally similar compounds, making it difficult to interpret their product-ion spectra. Electron transfer dissociation (ETD), a relatively new technique for protein and peptide fragmentation, has been shown to provide more detailed structural information. In this study, we compared the ability of CID with that of ETD to differentiate between eight 3,20-dioxosteroids that had been derivatizated with a quaternary ammonium salt, Girard reagent P (GirP), at room temperature or after exposure to microwave irradiation to generate doubly charged ions. We found that the derivatization of steroid with GirP hydrazine occurred in less than 10 min when the reaction was carried out in the presence of microwave irradiation compared to 30 min when the reaction was carried out at room temperature. According to the MS/MS spectra, CID provided rich, structurally informative ions; however, the spectra were complex, thereby complicating the peak assignment. In contrast, ETD generated simpler spectra, making it easier to recognize individual peaks. Remarkably, both CID and ETD were allowed to differentiate of steroid isomers, 17α-hydroxyprogesterone (17OHP) and deoxycorticosterone (DOC), but the signature ions obtained from CID were less intense than those generated by ETD, which generated much clearer spectra. These results indicate that ETD in conjunction with CID can provide more structural information for precise characterization of steroids.