Croton tiglium L. belongs to the family Euphorbiaceae, distributed mainly in countries like India and China. The seeds of C. tiglium in raw form exert toxic effects, and the purified seeds are used in medicinal formulations, especially in Ayurveda. The present study is aimed to evaluate the chemical changes of raw and purified samples of C. tiglium seeds using chromatographic techniques such as high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). HPTLC profiles of various extracts of raw and purified samples were developed to evaluate the chemical changes occurring during the purification process. Different extracts of raw and purified samples were screened using different derivatizing reagents to understand the nature of chemical compounds. HPLC technique was also employed for the comparative evaluation of various extracts. The phenolic and flavonoids contents of the purified sample were found to be less when compared to those of the raw sample. The HPTLC profiling of various extracts showed fractional removal of some acidic compounds, steroids, terpenoids, phorbol esters and coumarins in the purified sample. The chromatographic profiling of different extracts also exposed the differences in the chemical constituents of raw and purified samples. Removal of certain acidic compounds and phorbol esters was confirmed also by HPLC. The present study is the first attempt to monitor the chemical changes occurring during the purification process of C. tiglium seed using HPTLC, and the result indicated that purification imparted the chemical changes to certain chemical constituents.