An ultra performance liquid chromatography-triple quadrupole tandem mass spectrometric(UPLC-MS/MS) method was developed for the determination of α-lactalbumin(α-La) and β-lactoglobulin(β-Lg) in whey powder under positive ion multiple reaction monitoring(MRM) mode.The samples were firstly alkylated with iodoacetamide,followed by enzymatic hydrolysis with alkaline trypsin,then separated on a Waters ACQUITY UPLC-BEH C18,300 - column with 0.1% formic acid water solution and 0.1% formic acid acetonitrile solution as mobile phases by gradient elution.Results showed that there were good linear relationships for α-La and β-Lg in the ranges of 40-1 000 nmol/L and 80-2 000 nmol/L,respectively.The limits of quantitation(S/N=10) for α-La and β-Lg were 0.020 g/100 g,respectively.The average recoveries of two analytes from spiked whey powder samples were in the range of 84.7%-95.6%,with relative standard deviations(RSDs,n=6) of 1.6%-5.8%.Based on the above method,the targets in four kinds of whey powder samples,i.e.70% demineralised whey powder,90% demineralised whey powder,whey protein concentrate and α-lactalbumin powder from five countries were determined and contrasted.With the advantages of simple operation,accuracy and reliability,the established method was suitable for the determination of α-La and β-Lg in whey powder.