Protein O-GlcNAcylation is a prevalent and dynamic post-translational modification that targets a multitude of nuclear and cytoplasmic proteins. Through the modification of diverse substrates, O-GlcNAcylation plays a pivotal role in essential cellular processes, including transcription, translation, and protein homeostasis. Dysregulation of O-GlcNAc homeostasis has been implicated in a variety of diseases, including cardiovascular diseases, cancer, and neurodegenerative diseases. Studying O-GlcNAcylated proteins in different tissues is crucial to understanding the pathogenesis of these diseases. However, identifying phenotype-relevant candidate substrates in a tissue-specific manner remains unfeasible. We developed a novel tool for the analysis of O-GlcNAcylated proteins, combining a catalytically inactive CpOGA mutant CpOGA(CD) and TurboID proximity labeling technology. This tool converts O-GlcNAc modifications into biotin labeling, enabling the enrichment and mass spectrometry (MS) identification of O-GlcNAcylated proteins in specific tissues. Meanwhile, TurboID-CpOGA(DM), which carries two point mutations that inactivate both its catalytic and binding activities toward O-GlcNAc modification, was used as a control to differentiate O-GlcNAc-independent protein-protein interactions. We have successfully used TurboID-CpOGA(CD/DM) (TurboID-CpOGA(M)) to enrich O-GlcNAc proteins in Drosophila combining the UAS/Gal4 system. Our protocol provides a comprehensive workflow for tissue-specific enrichment of candidate O-GlcNAcylated substrates and offers a valuable tool for dissecting tissue-specific O-GlcNAcylation functions in Drosophila. Key features • Innovative approach to studying O-GlcNAcylation: Combines a catalytically inactive CpOGA mutant (CpOGA(CD)), TurboID proximity labeling technology, and the UAS/Gal4 system for tissue-specific analysis. • Tissue-specific focus: Enables enrichment and mass spectrometry (MS) identification of O-GlcNAcylated proteins in specific tissues of Drosophila. • Biotin labeling conversion: Converts O-GlcNAc modifications into biotin tags, facilitating downstream enrichment and analysis. • Powerful tool for understanding the role of O-GlcNAcylation in cellular processes and its involvement in diseases such as cardiovascular diseases, cancer, and neurodegenerative disorders.