Hydrophilic vitamins play an important role in human health. The analysis of these compounds is indispensable for monitoring their content in pharmaceuticals and food in order to prevent some human diseases. The separation and identification of eight hydrophilic vitamins, i.e., B 1 , B 2 , B 3 , B 5 , B 6 , B 9 , B 12 , and C, was achieved by thin layer chromatography and Raman spectrometry. After chromatographic experiments, the best results were obtained using programmed multiple development. The unambiguous identification of superimposed spots can be done without using standards of vitamins.