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One of the distinctive characteristics of cacti species is the presence of alkaloids. Alkaloids are nitrogenated molecules with hallucinogenic and pharmacological properties in humans and other animals. Plant cell, tissue, and organ culture have emerged as an effective tool for investigating the biosynthesis of a variety of functional metabolites and for studying the preservation of endangered plant species. In this study, we examined the alkaloid and nitrogenated compound profiles of the aerial and radicular sections of Coryphantha macromeris plants that were cultivated in both greenhouse and in vitro conditions. Additionally, we analyzed the callus cultures generated from stem discs. To perform these analyses, Ultra-High-Performance Liquid Chromatography coupled with High-Resolution Mass Spectrometry (UHPLC-PDA-HESI-Orbitrap-MS/MS) was utilized. Under the working parameters, 78 compounds were detected, and 61 of them were identified. Among the identified compounds, the in vitro plants presented 24 compounds, greenhouse plants a total of 21 compounds, and callus tissue 16 compounds. On the other hand, 7 compounds (laurydiethanolamine, toluic acids, and their derivatives) were detected in all systems, suggesting that these metabolites may serve as markers to help find the authenticity of C. macromeris preparations, and that, plant and cell-tissue cultures with this plant species are suitable for the biosynthesis of the selected compounds. In addition, our research suggests that no alkaloids with reported psychotropic properties are present in C. macromeris.