Gloriosine, the predominant metabolite of Gloriosa superba L., shares chemical properties with colchicine. We analyze the microtubule-binding affinity of gloriosine at the colchicine binding site (CBS) using an in silico-in vivo approach. The In silico docking of gloriosine showed a binding score of (-) 7.5 kcal/Mol towards beta-tubulin at CBS and was validated by overlapping the coupling pose of the docked ligand with co-crystallized colchicine. 2D plots (Ligplot +) showed > 85% overlap between gloriosine and colchicine. The ADMET profile of gloriosine was in accordance with Lipinski's rule of five. Gloriosine belongs to class II toxicity with anLD(50) value of 6 mg/kg. In vivo and transmission electron microscopy studies revealed that gloriosine induces abnormalities in cell division such as condensed chromosomes in C-metaphase and enlarged nucleus with increased nuclear material. Gloriosine treated cells exhibited mitotic index of about 14% compared to control of 24% and high anti-proliferative activity i.e. 63.94% cell viability at a low concentration (0.0004 mg/ml). We conclude that gloriosine has a strong affinity for beta-tubulin at CBS and thus can be used as a colchicine alternative in cytology and other clinical conditions. CI - (c) 2023. The Author(s).