Cannabinoid synthases are flavoenzymes that catalyse the production of cannabinoids in the plant Cannabis sativa. Cannabinoid synthases are classified based on the major cannabinoid produced, as either cannabidiolic acid synthase (CBDAS), tetrahydrocannabinolic acid synthase (THCAS), or cannabichromenic acid synthase (CBCAS). Due to the medicinal importance of these specialized metabolites, heterologous hosts expressing cannabinoid synthases have been engineered as microbial cell factories for production of cannabinoids. Production of active recombinant cannabinoid synthases is dependent on correct folding of their complex structural architecture, which is controlled by glycosylation of select protein residues. They share >90% amino acid sequence similarity with at least seven conserved glycosylation sites identified previously. Glycosylation site analysis has been performed on THCAS, but only limited information exists for CBDAS and CBCAS enzymes. In this study, we set out to document the glycosylation status of the three different cannabinoid synthases when produced in the heterologous host Pichia pastoris. To this end, we expressed the three cannabinoid synthases as secreted products followed by strong cation exchange chromatography-based purification. The proteins were then subjected to trypsin digestion and EndoH-based deglycosylation. Glycopeptides were enriched using Hydrophilic Interaction LIquid Chromatography (HILIC)-based and analyzed using liquid chromatography – mass spectrometry (LC-MS/MS) and bioinformatics. We identified several glycopeptides for each enzyme. Analysis of these glycopeptides showed that the glycosylation was predominantly N-linked. A proportion of the observed glycosylated residues correlated with those documented in literature. In addition, we have also observed peptides with O-linked glycosylation. We also determined the enzymatic activity and structural stability of the glycosylated enzymes using LC-MS based enzyme assay and thermoflour assay respectively. In this study, we aim to correlate the glycosylation status of cannabinoid synthases under heterologous conditions with their enzymatic function. We anticipate this will improve the understanding of recombinant cannabinoid synthase structure-function relationship and provide additional information prior to site-directed mutagenesis for efficient production of specialized metabolites