Autophagy is a highly conserved catabolic process in eukaryotic cells. Reactive nitrogen species play roles as inductors and signaling molecules of autophagy. A key mechanism of NO-mediated signaling is S-nitrosylation, a posttranslational modification (PTM) of proteins at cysteine resi-dues. In the present work we analyzed the patterns of protein S-nitrosylation during the induc-tion of autophagy in Triticum aestivum roots. The accumulation of S-nitrosylated proteins in the cells during autophagy induced by KNO2 and antimycin A was visualized using monoclonal an-tibodies by Western blot analysis, and proteins were identified using a standard bottom-up pro-teomics approach. Protein S-nitrosylation is a labile and reversible PTM, and therefore SNO group can be lost during experimental procedures. Subsequent bioinformatic analysis using predictive algorithms and protein-ligand docking showed that identified proteins possess hypothetical S-nitrosylation sites. Analyzing protein-protein interaction networks enabled us to discover the targets that can directly interact with autophagic proteins, and those that can interact with them indirectly via key multifunctional regulatory proteins. In this study, we show that S-nitrosylation is a key mechanism of NO-mediated regulation of autophagy in wheat roots. A combination of in silico predictive algorithms with a mass spectrometry analysis provides a targeted approach for the identification of S-nitrosylated proteins.