Gangliosides are ubiquitously present on cell surface. They are more abundantly expressed in nerve cells and tissues and involved in pathology of various diseases. Diversity of molecular structures in the carbohydrate head group, fatty acyl, and long chain base increases the complexity of analyzing gangliosides. In this study, an ultrahigh-performance liquid chromatography-tandem mass spectrometry method is developed for analysis of the co-eluting ganglioside isomers, which uses ion polarity switching to integrate glycan head isomer identification, ceramide isomer differentiation, and quantification of ganglioside into one analysis. The method is facilitated with an extensive ganglioside target list by combining the various glycan head groups, long chain bases, and the experimentally determined fatty acyls. Correlation between the retention time of ganglioside and its ceramide total carbon number is experimentally validated and used to predict retention time of ganglioside target list for scheduling the final multiple reaction monitoring method. This method was validated according to the FDA guidelines: 96.5% of gangliosides with good accuracy (80-120%), precision (< 15%), and linearity R > 0.99. The authenticated gangliosides were quantified from mouse brain by isotope dilution. Overall, 165 gangliosides were quantified using 10 mg mouse brain tissue, including 100 isomers of GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, and GT1b.