The separation of 14 water-soluble vitamins and vitamin cofactors was investigated by micellar electrokinetic capillary chromatography and diode array detection using sodium cholate as the micellar phase. The method was optimised with respect to the effect of buffer composition, capillary temperature and applied voltage resulting in separation of all compounds in about 25 min. With the current method it is possible to predict the eluting order of the individual compounds from their net charges of each compound because of a low ion pairing between solutes and micelles. The linearities within concentration ranges of up to two-orders of magnitudes were good with correlation coefficients from 0.971 to 0.997. The separation efficiency was satisfactory with a good resolution ranging from 2 to 45 and a theoretical number of plates varying from 200,000 to 480,000. The repeatability of the developed method showed relative standard deviations on migration time in the range from 0.5% to 1.2% (n = 15) and for normalised peak areas, relative standard deviations were approximately 6%.