In the present study, to systematically investigate the post-translational regulatory mechanisms of IRF3 (Interferon Regulatory Factor 3), we conducted comprehensive protein-binding mass spectrometry analysis combined with modification-specific mass spectrometry profiling targeting this key transcription factor. The primary objectives of these experimental approaches were twofold: first, to identify and characterize the potential types of post-translational modifications (PTMs) that IRF3 may undergo—including but not limited to phosphorylation, ubiquitination, acetylation, and sumoylation—and second, to screen for candidate enzymes that are likely to mediate or regulate these modifications, such as kinases, ubiquitin ligases, deacetylases, or SUMO E3 ligases. By integrating data from both protein-protein interaction and PTM profiling, this study aims to provide novel insights into the molecular machinery underlying IRF3’s functional regulation, thereby laying a foundation for further elucidating its role in innate immunity, inflammatory responses, and other pathophysiological processes.