Niemann-Pick Disease Type C (NPCD) is a rare neurodegenerative disorder affecting 1 in 120,000 live births, characterized by complex alterations in sphingolipid metabolism. While cholesterol accumulation is a prominent feature, the situation is more intricate in the brain, where primary accumulating lipids are sphingolipids such as GM2 and GM3 gangliosides. So far, the only approved therapy to date, Miglustat, targets sphingolipid pathways and is used to prevent disease progression. The objective of this study is to improve an existing ganglioside assay for its utilization in cellular models. The optimization process encompasses the careful selection of the most effective extraction buffers, optimizing the protocol steps to enhance efficiency, and selecting appropriate sample sizes for cellular pellets. This study aims to develop a universally applicable assay that can be used across a wide range of cell lines. The assay validation involves the utilization of differentiated astrocytes and neural progenitor cells derived from fibroblast lines harboring NPC1-heterozygous and NPC1-homozygous variants. Quantification of gangliosides is performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS).The findings of this investigation demonstrated a notable disparity in the buildup of GD3 ganglioside in fibroblast cell lines that were homozygous for the NPC1 gene mutation. Conversely, neural progenitor cells and astrocytes exhibited a reduction in GD3 ganglioside levels. Furthermore, it was observed that NPC1-homozygous fibroblasts exhibited increased concentrations of complex GD1a and GT1b gangliosides. Additional investigation is required to effectively address the inquiries that have been prompted by these results. This assay holds significant potential, hence redefining the application of cellular models in in vitro experiments related to lysosomal storage diseases. Upon optimization, the ganglioside assay will function as a valuable instrument for capturing the accumulation pattern of gangliosides that are linked to rare diseases.