Fragile X syndrome (FXS) is an X-linked neurodevelopmental condition associated with intellectual disability and behavioral problems due to the lack of the Fragile X mental retardation protein (FMRP), which plays a crucial role in synaptic plasticity and memory. A desirable in vitro cell model to study FXS would be one that can be generated by simple isolation and culture method from a collection of a non-invasive donor specimen. Currently, the various donor-specific cells can be isolated mainly from peripheral blood and skin biopsy. However, they are somewhat invasive methods for establishing cell lines from the primary subject material. In this study, we characterized a cost-effective and straightforward method to derive epithelial cell lines from urine samples collected from participants with FXS and healthy controls (TD). The urine-derived cells expressed epithelial cell surface markers via fluorescence-activated cell sorting (FACS). We observed inter, and the intra-tissue CGG mosaicism in the PBMCs and the urine-derived cells from participants with FXS potentially related to the observed variations in the phenotypic and clinical presentation FXS. We characterized these urine-derived epithelial cells for FMR1 mRNA and FMRP expression and observed some expression in the lines derived from full mutation mosaic participants. Further, FMRP expression was localized in the cytoplasm of the urine-derived epithelial cells of healthy controls. Deficient FMRP expression was also observed in mosaic males, while, as expected, no expression was observed in cells derived from participants with a hypermethylated full mutation.