Novel modifications were applied to high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) for the separation and quantitation of the steroidal glycoalkaloids (SGAs) solanine, chaconine, solamargine, and solasonine as well as the steroidal glycoalkaloid aglycones (SGAAs) solasodine and solanidine. Because attempts to develop a gradient elution HPLC method were only marginally successfully and non-robust, it was deemed more practical to develop separate HPLC methods for either the SGAs or SGAAs of interest. Furthermore, a novel approach using methanol as a mobile phase modifier was still required to successfully separate solamargine and chaconine. Comparing potential mobile phase buffers, ammonium dihydrogen phosphate was chosen as the most efficient, stable, and economical. Separations were best realized isocratically at a column temperature of 50 °C for the SGAs and either 26 °C or 50 °C for the SGAAs. Progesterone was applied as an internal standard. Effects of pH were also tested. Figures of merit such as limit of detection (LOD), limit of quantitation (LOQ), and linear dynamic range are described herein. Lastly, solid-phase microextraction (SPME) using on-fiber derivatization coupled with GC-MS was investigated for extraction and analysis of these SGAAs. A carbowax divinylbenzene (CW-DVB) coated SPME fiber was the most suitable. Solanidine could be extracted and identified directly using our SPME/GC-MS method while solasodine required a derivatization step involving trimethylsilylimidazole (TMSI). Although initial attempts were qualitatively reproducible, eventual degradation to fibers precluded complete study. Cholesterol as an internal standard was investigated.