A new solvent-free method for the simultaneous determination of some major phytoestrogens (equol, enterodiol, daidzein, genistein, glycitein) in different commercial milks (cow, goat and soy-rice) was developed. After solid phase microextraction, performed by direct immersion of a 65 μm-polydimethylsiloxane-divinylbenzene fiber in diluted (1:100 with 0.2% formic acid - 30% sodium chloride) milk samples (18 °C for 20 min under stirring), a direct on-fiber silylation with N,O-bis (trimethylsilyl)trifluoroacetamide) containing 1% trimethylchlorosilane (70 °C for 20 min) was performed prior to gas chromatography-mass spectrometry analysis. Since the target compounds were determined as aglycones, the hydrolytic removal of the aglycone from the glycosides was performed. The method permitted the determination of the target analytes in all the considered milk samples as well as the detection of some major amphipathic fats indicating that the approach could potentially be applied in the future for further applications, such as milk profiling.