Abstract: An auspicious method for the quantitative determination of vitamin A (retinol) was augmented in seven different Jordanian local fresh milk samples using liquid chromatography tandem -mass spectrometry (LC/MS/MS). The compound was separated with C18 Thermo fisher gold (4.50 x100 mm x 5 Å) column through a flow rate of 1 mL/min with isocratic mobile phase. Vitamin A acetate was used as an internal standard .The method was validated using triplicate analyses, relative recovery experiment and statistical analysis. Liquid- liquid extraction was employed as a pre-concentration step with n-hexane -dichloromethane mixture (90%:10%) as an extraction solvent.