A sensitive liquid chromatography/electrospray ionization-tandem mass spectrometry method for the determination of free and conjugated estrogens in cow milk is described. Milk samples were diluted with water, then extracted and cleaned-up by solid-phase extraction using graphitized carbon black as adsorbent material, without any enzymatic cleavage, derivatization and/or protein precipitation step. Two fractions were collected (free and conjugated estrogens) and analyzed separately. Mass spectrometry analysis was performed in negative ion mode using selected reaction monitoring acquisition mode. For all compounds the coefficients of determination R2 ranged between 0.9892 and 0.9997. Analytical recoveries were in the range of 86-109% for free estrogens and 85-118% for conjugates, with relative standard deviations below 10%, and method detection limit ranged between 3 and 80 ng L-1. Finally, the developed method was used to determine the presence of free and conjugated estrogens in six retail milk samples (five cow milk samples and one goat milk sample) and one goat milk sample provided by a local shepherd. Estrone was found to be the major free estrogen present in commercial milk samples, and estrone 3-sulfate showed the highest concentration among the target conjugated estrogens. Estriol was also observed in some analyzed milk samples, but the concentrations were always below the limit of quantification.