Hexahydrocannabinol (HHC) is a semi-synthetic drug structurally related to natural cannabinoids, manufactured from hemp-extracts. By cyclization and catalytic hydrogenation, cannabidiol (CBD) is converted into a mixture of (9R)-HHC and (9S)-HHC. The (9R)-HHC epimer has THC-like psychoactive effects. HHC was first reported to EMCDDA in August 2022. By April 2023, 20 EU Member States have detected the substance. Human metabolism studies and analytical methods for investigating HHC metabolites in human urine are lacking. This study aims (I) to present an analytical method for screening HHC metabolites in human urine; (II) to investigate the Phase I HHC metabolites by performing in vitro and in vivo metabolite identification studies of human liver microsome (pHLM), S9 fraction (pS9) and urine samples; (III) and to present a quantitative confirmatory method optimised by the investigation of authentic urine samples.