Thin-layer chromatographic procedures are described for the analysis and separation of a variety of organic molecules (amino acids, B-complex vitamins, purines and pyrimidines, neutral lipids, polar sugars and dyes) using water-in-oil-microemulsion containing N-cetyl-N, N, N trimethyl ammonium bromide (CTAB) as mobile phase. High performance thin layer chromatographic plates of silica gel G and cellulose were used as stationary phases. The proposed method is suitable for selective separation of bromocresole green from other dyes on cellulose HPTLC plates. Furthermore, vitamins (thiamine, pyridoxine and riboflavin in marketed formulations) were successfully resolved on silica HPTLC plates. Effect of other biomolecules as impurities was investigated on simultaneous separation of thiamine, pyridoxine riboflavin from their mixtures. The well intense colored compact spots for all compounds under study were realized.