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A method of ultra-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UPLC-ESI-MS/MS) has been established for simultaneous determination of major disialoganglioside 3 (GD3) and monosialoganglioside 3 (GM3) in infant formulas and whey protein concentrates. Gangliosides were extracted by using the technique of Svennerholm and Fredman and then cleaned up with OASIS HLB solid-phase extraction (SPE) cartridges. The various molecular species of gangliosides were separated on an Acquity UPLC BEH C8 column and analyzed under the negative ion mode. GD3 and GM3 were rapidly quantified using internal standard (IS) method. The developed method was further validated by determining the linearity, average recovery, sensitivity (limit of quantification), and precision. The results presented high correlation coefficients (R2 > 0.993) of the selected 16 gangliosides molecular species and provided the respective linear ranges. The limit of quantification was 0.3250.734 mg/100 g for eight molecular species of GD3 and 0.0080.312 mg/100 g for eight molecular species of GM3, respectively. The reasonable average recoveries (8195%) and precision (relative standard deviation [RSD] =15%) were also demonstrated in three different spiked levels. This new method would be very useful in the quantitative determination of gangliosides in infant formulas and whey protein concentrates.