Lessertia frutescens L., commonly known as cancer-bush, is a medicinally reputed plant species indigenous to southern Africa. Field leaf extracts of this species are known to exhibit many curative properties. However, little is known about the bioactive compounds that are present in in vitro leaf extracts and seed extracts. The objective of this study was to verify the presence of and quantify L-canavanine, gamma amino butyric acid (GABA), arginine and D-pinitol in the seeds, field leaves and in vitro leaves of L. frutescens using gas and liquid chromatography. Methanolic extracts of in vitro leaves, field leaves and seeds were used. MRM chromatograms were recorded for L-canavanine and arginine using tandem mass spectrometry. GC chromatograms were recorded for GABA and D-pinitol using gas chromatography. D-Pinitol was found to be most abundant and was 14.75 and 18.17 mg/g in in vitro and field leaf extracts respectively, followed by GABA (7.29 and 3.48 mg/g), arginine (7.08 and 0.35 mg/g) and L-canavanine (0.55 and 0.08 mg/g). In the seed extracts, GABA content was found to be the highest (1.69 mg/g) followed by L-canavanine (0.37 mg/g), then D-pinitol (0.25 mg/g), and arginine (0.02 mg/g). In vitro leaves had higher quantities of all compounds, except for D-pinitol. This study therefore highlights the potential of bulking in vitro leaves for the extraction of the medicinal compounds, L-canavanine and GABA.