Vitamins are an heterogenous family of drugs with very different molecular properties. A novel method for the simultaneous quantification by LC-tandem mass spectrometry of ten water- and fat-soluble vitamins (retinol, α-tocopherol, thiamin, riboflavin, nicotinamide, pantothenic acid, pyridoxine, biotin, folic acid and ascorbic acid) in parenteral admixture without sample pre-treatment is described in this paper. The separation was achieved by reversed phase LC with a Zorbax C18 column (length 150 mm, internal diameter 4.6 mm, particle size 2.5 μm). A gradient of mobile phase was set up starting at 100% ammonium acetate solution at 10 mM, pH = 4.5 reaching a step of ammonium acetate : methanol in 15 min (65:35 v/v). The detection system combined online photodiode array and tandem mass spectrometry. Degradation studies of vitamin admixtures were performed and the method proved to be stability indicating. The LC-UV-MS-MS method was validated in accordance with the ICH guidelines. Repeatability and intermediate precision were comprised between 1.0 and 10.2% and 0.7 and 11.8%, respectively. A stability study of the vitamins mixed within three different parenteral nutrition admixtures has been performed and has shown their stability within at least 48 h.