Background: The genus Gloriosa is commercially valued due to its colchicine metabolite, which is clinically used in gout and as an antimitotic agent. Objectives: The study was a comparative pharmacognostical evaluation and simultaneous high performance thin layer chromatography (HPTLC) quantification of bioactive alkaloids in G. superba, G. lutea and G. rothschildiana. In vitro anti-gout activity was also established. Materials and Methods: Pharmacognostical studies and metabolic variations were analyzed per the Ayurvedic Pharmacopoeia of India and validated HPTLC method. Inhibition of protein denaturation, hydroxyl radical scavenging and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay evaluated the anti-gout activity. Results: The morpho-anatomical studies suggest that the target species are similar with no characteristic difference, apart from the flower colour. The pharmacognostic standards were also established as per the Ayurvedic Pharmacopoeia of India to ensure the quality of raw material. Quantification of colchicine and gloriosine content through validated HPTLC method reveals significant variation, ranging from 0.046% to 0.860%, and from 0.040% to 0.198% on dry wt. basis. The maximum content of both the targeted metabolites was in G. superba, followed by G. lutea and G. rothschildiana. The highest in vitro anti-gout and radical scavenging activity among the three species was in G. superba. Conclusion: The pharmacognostic standards of three Gloriosa species were established, and this opens avenues for chemotaxonomic studies on G. lutea and G. rothschildiana from different phytogeographical zones of India for the identification of their elite germplasm. The study also led to the identification of alternate species of G. superba which can be explored commercially to meet the industrial demand for colchicine.