A liquid chromatographic (LC) method was established for the determination of α-casein, β-casein, κ-casein, α-lactalbumin and β-lactoglobulin in ultra high temperature (UHT) milk. The proteins in milk were denatured by adding a buffer solution containing bis[tris(hydroxymethyl)aminomethane (BisTris), guanidine hydrochloride, sodium citrate, dithiothreitol, etc. The analytes were detected by an ultraviolet (UV) detector and quantified by the external standard method. The results showed that the calibration curve of each protein had good linearity with a correlation coefficient above 0.995. The recoveries of spiked samples were between 95.2% and 105.0%, the detection limit was 0.01–0.02 g/100 mL, and the relative standard deviation was 0.78%–2.70%. The method is characterized by good separation effect, good repeatability and simple operation, and is suitable for quantitative analysis of α-casein, β-casein, κ-casein, α-lactalbumin and β-lactoglobulin in UHT milk.