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In the literature, there is a scarcity of greener analytical approaches for colchicine (CLH) analysis. As a result, efforts were made in this study to develop and validate a greener reversed-phase high-performance thin-layer chromatography (HPTLC) technique for CLH analysis in traditional extracts (TE) and ultrasonication-based extracts (UBE) of commercial Unani formulations, commercial allopathic formulations, and Colchicum autumnale Pleniflorum (L.) obtained from Egypt and India. This new technique was compared to the regular normal-phase HPTLC method. The greenness profile of both methods was estimated using the Analytical GREENness (AGREE) approach. In the 100–600 and 25–1200 ng/band ranges, regular and greener HPTLC procedures were linear for CLH analysis, respectively. For CLH analysis, the greener HPTLC method was more sensitive, accurate, precise, and robust than the regular HPTLC method. For CLH analysis in TE and UBE of commercial Unani formulations, commercial allopathic formulations, and C. autumnale obtained from Egypt and India, the greener HPTLC method was superior in terms of CLH content compared to the regular HPTLC method. In addition, the UBE procedure was superior to the TE procedure for both methods. The AGREE scores for regular and greener reversed-phase HPTLC methods were found to be 0.46 and 0.75, respectively. The AGREE results showed excellent greener profile of the greener HPTLC method over the regular HPTLC technique. Based on several validation criteria and pharmaceutical assay findings, the greener HPTLC method is regarded as superior to the regular HPTLC approach. Full article