Callus was induced from different explants of Gloriosa superba L. on MS medium supplemented with different concentrations of 2,4- D. Cell suspension cultures derived from callus of Gloriosa superba L. were established quantified for the colchicine content using High Performance Liquid Chromatography. Effect of time on the growth of cells and colchicine production by cell suspension cultures in liquid medium was also studied. Maximum growth index (0.331 ± 0.0016) was obtained after four weeks of cell growth. Itwas observed that the colchicine yield reached 13.33 ± 0.024 μg/g dry wt. in cells and 59.30 ± 0.54 μg/l in residual liquid medium after four weeks of culture in liquid growth medium and thereafter there was reduction in biomass as well as colchicine content.