BACKGROUND: Psilocybin-containing mushrooms are gaining the attention of the scientific community due to the potential benefits offered by their psychoactive phytochemicals in the treatment of addiction and various mental health conditions. Although there are hundreds of different Psilocybe species, only a handful have been successfully cultivated under indoor controlled conditions and chemically analyzed. OBJECTIVE: The goal of this publication is to describe Nammex's ongoing effort to cultivate poorly studied Psilocybe mushroom species and analyze them by High Performance Thin-Layer Chromatography (HPTLC) to identify and quantify important psychoactive compounds. METHODS: Pure mycelium cultures of Psilocybe species were created from spore prints and tissue of mushrooms collected in the wild. From these mycelia, numerous cultivars were developed and then propagated on various substrates, based upon nutritionally supplemented cellulosic materials. Using indoor growth chambers under strictly controlled conditions, mushrooms were produced and prepared for analysis. RESULTS: Six Psilocybe species (P. zapotecorum, P. natalensis, P. azurescens, P. subaeruginosa, P. cyanescens, and P. stuntzii) were successfully cultivated indoors. Species identity was confirmed through analysis of anatomical and microscopic features, as well as by DNA sequencing. HPTLC was successfully used to quantify psilocybin and psilocin and identify norbaeocystin, baeocystin, and aeruginascin. P. zapotecorum had the highest psilocybin content (1.89%) and P. stuntzii the lowest (0.45%). Preliminary data showed that psilocybin concentrations remained stable across three successive flushes of P. stuntzii. Storage of fresh mushrooms in -20 °C freezer prior to freeze drying drastically reduced psilocybin and increased psilocin levels. CONCLUSIONS: This study successfully demonstrated the cultivation and chemical profiling of multiple Psilocybe species under controlled conditions. The detailed HPTLC analysis revealed species-specific differences in psychoactive compound concentrations. Future research will incorporate advanced techniques, such as HPLC and mass spectrometry, to develop a more comprehensive chemical profile of these mushrooms.