Background: Niemann–Pick type C is a lysosomal storage disorder that results from pathogenic variants in the NPC1 gene or in some cases from NPC2 pathogenic alterations. The disease presents a remarkable clinical variability that in some cases resembles common diseases, often resulting in a diagnostic odyssey or at least delaying proper diagnosis. In addition, the NPC1 gene is highly polymorphic, and consequently, when missense variants are identified after gene sequencing, accurate classification of their pathogenicity is essential to ensure appropriate access to available therapies and to provide reliable genetic counseling. Objectives: To get insights into the pathogenicity of a novel variant in NPC1, p.Cys800Ser, we created stable cell lines expressing this variant, in parallel with cell lines expressing the NPC1 wild-type and NPC1 pathogenic variants. Methods: We leveraged an isogenic cell line in which the NPC1 gene was knocked down and subsequently infected it with retroviruses carrying NPC1-WT and NPC1 variants C-terminally fused with an mNeonGreen tag. Three different NPC1 variants were included in this study: two known pathogenic variants, p.Ala1035Val and p.Pro1007Ala, and the novel p.Cys800Ser, whose significance was unknown. Results: We observed in the stable cell line expressing NPC1 p.Cys800Ser that the mutated NPC1 protein is transported to the lysosome similarly to the p.Pro1007Ala variant and affects lysosomal distribution. Conclusions: Using this approach, we could analyze the pathogenicity of each variant separately and these cell lines could be used for personalized medicine-based approaches and multi-omic studies.