A High performance thin layer chromatography (HPTLC) method was developed and optimized for determination of colchatetralene, a new bioactive alkaloid isolated from the chloroform extract of an endophytic fungus, isolated from surface sterilized seeds of Gloriosa superba Linn. The development of method involved evaluation and optimization of the various stages of sample preparation, chromatographic separation, detection and quantification. The method employed aluminum backed pre coated TLC plates (silica gel 60F254) as the stationary phase. The mobile phase system consisted of ethyl acetate-methanol-water (10:1.5:1, v/v/v) at room temperature (25 ± 2°C). Densitometric analysis was carried out in the absorbance/reflectance mode at the wavelength of 236nm. The system was found to give compact spots for colchatetralene (Rf value of 0.36 ± 0.03). The linear regression analysis data for the calibration plots showed good linearity (r2 = 0.99601 ± 0.002) in the concentration range 200–1200 ng spot-1. The method was validated according to the International Conference on Harmonization (ICH) guidelines for precision, accuracy, recovery and robustness. The limits of detection and quantification were 20 and 60 ng per spot, respectively. © 2013 Bentham Science Publishers.