Reversed phase thin layer chromatography (RPTLC) of non-ionic and cationic surfactants was performed on silica gel layers impregnated with 5% paraffin oil, silicone oil and tributyl phosphate as stationary phases using water and organic polar solvents as mobile phases. In all, four stationary phases and twelve mobile phases were used to examine the mobility pattern and to find out best TLC system for separation of surfactants from their mixtures. The TLC system comprising of 5% paraffin oil impregnated silica gel layers as stationary phase and pure methanol as mobile phase was found most favourable for achieving separation of non-ionic surfactants (Tx-100, Brij-35 and Tween-20) from cationic surfactants (CPC, CTAB and TTAB). The effects of loading amount of analyte and metal cations impurities in the sample have been examined on mutual separation of Tx-100 and TTAB. The results obtained with methanol were compared with those obtained with ethanol and butanol. The mobility of surfactants was found to increase with the increase of polarity of the alcohol used as mobile phase.