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Summary O-linked β-N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification involved in a wide range of signaling pathways, but its specific role in regulating biological processes remains unclear. This protocol describes approaches to understand O-GlcNAc’s role in fibroblast contraction. Specifically, cellular O-GlcNAc levels are controlled through treatment of fibroblasts with inhibitors in both 2D and 3D cultures. We then describe 2D contraction assay and 3D collagen gel contraction assay to analyze the effect of the modification on sphingosine-1-phosphate signaling for contraction. For complete details on the use and execution of this protocol, please refer to Pedowitz et al. (2021) .