Plant cell, tissue, and organ culture have become a powerful technology for the production of biomass, and for the research and biosynthesis of a variety of secondary metabolites. In this work, we report an efficient method for friable callus induction applied to the medicinal cactus Coryphantha macromeris, its kinetic behavior, and its phytochemical profile, assessed at the maximum biomass production phase. Callus cultures were obtained from stem discs inoculated on Murashige and Skoog medium (MS) supplemented with 6-benzylaminopurine (BAP; 2.2 µM) and picloram (4.14 µM). The highest biomass production (20.65 g DW L−1) was achieved at nine weeks of culture. Then, the phytochemical profile was analyzed using Ultra-High-Performance Liquid Chromatography-tandem Mass Spectrometry (UHPLC-PDA-HESI-Orbitrap-MS/MS). Chromatographic and mass spectrometric analyses indicated the presence of 61 metabolites, with 52 being identified. Among these compounds, 11 organic acids, 16 phenolic acids, 8 flavonoids, and 17 metabolites of different classes were identified. Our results could significantly contribute to the current knowledge of tissue culture of cacti species, as well as the potential applications of the in vitro callus culture of C. macromeris. Furthermore, we report the presence of some metabolites in cell culture of cacti species and their fragmentation pattern for the first time.