IntroductionEmpirical research has refined traditional herbal medicinal systems. The traditional market is expanding globally, but inadequate regulatory guidelines, taxonomic knowledge, and resources are causing herbal product adulteration. With the widespread adoption of barcoding and next-generation sequencing, metabarcoding is emerging as a potential tool for detecting labeled and unlabeled plant species in herbal products.MethodsThis study validated newly designedrbcLandITS2metabarcode primers for metabarcoding using in-house mock controls of medicinal plant gDNA pools and biomass pools. The applicability of the multi-barcode sequencing approach was evaluated on 17 single drugs and 15 polyherbal formulations procured from the Indian market.ResultsTherbcLmetabarcode demonstrated 86.7% and 71.7% detection efficiencies in gDNA plant pools and biomass mock controls, respectively, while theITS2metabarcode demonstrated 82.2% and 69.4%. In the gDNA plant pool and biomass pool mock controls, the cumulative detection efficiency increased by 100% and 90%, respectively. A 79% cumulative detection efficiency of both metabarcodes was observed in single drugs, while 76.3% was observed in polyherbal formulations. An average fidelity of 83.6% was observed for targeted plant species present within mock controls and in herbal formulations.DiscussionIn the present study, we achieved increasing cumulative detection efficiency by combining the high universality of therbcLlocus with the high-resolution power of theITS2locus in medicinal plants, which shows applicability of multilocus strategies in metabarcoding as a potential tool for the Pharmacovigilance of labeled and unlabeled plant species in herbal formulations.