The simultaneous quantification of 15 mycotoxins in cow milk by liquid chromatography-mass spectrometry, is presented. Extraction was performed with acidified acetonitrile, followed by a cleanup step with sodium acetate. During validation limits of detection (LOD) and quantification, linearity, precision, accuracy, recovery, matrix effect, and stability were studied. LOD values were between 0.02 and 10.14 ng/mL for aflatoxins M1, B1, B2, G1, G2, ochratoxins A and B, HT-2 and T-2 toxins, deepoxy-deoxynivalenol, zearalenone, sterigmatocystin and fumonisins B1, B2 and B3. Recovery values were between 82.6 and 94.4% for all the mycotoxins, except for fumonisins. The recovery values for fumonisins were between 42.1% and 64.6%. Matrix effect, between 25.5 and 96.8%, appeared for all of the mycotoxins, especially for deepoxy-deoxynivalenol, zearalenone and sterigmatocystin. The validated method achieves the quantification of those mycotoxins of major concern and mycotoxins that are not frequently studied in milk, such as fumonisins, sterigmatocystin or ochratoxin B.