A simple, precise and accurate high performance thin layer chromatography (HPTLC) method have been developed for the quantification of strychnine and brucine.  The strychnine and brucine are isolated from biological samples and herbal formulations using a liquid–liquid extraction procedure.  The clean-up procedure is performed using an acid solution.  The method has a linear range of 45 – 1000 ng/spot for strychnine and  95 – 1000 ng/spot for brucine.  The method precision was found to be 0.86 - 2.61 (% RSD) and 0.77 - 2.46 (% RSD) for strychnine and brucine respectively.  Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 101.34 % for strychnine and 101.23 % for brucine.