Protein arginine deiminases (PADs) convert arginine to citrulline, altering protein structure and function. Of the five human isozymes, PAD1–4 are catalytically active with distinct tissue-specificities, yet isozyme-specific substrate recognition remains poorly defined. Here, we provide a systematic, isozyme-wide comparison of human PAD1–4 by combining controlled in vitro incubation of human cell lysates with recombinant enzymes, deep-coverage LC–MS/MS and advanced computational validation. Importantly, by incubating lysates under uniform, saturating enzyme and calcium conditions we aimed to isolate intrinsic substrate recognition potential from confounding factors such as isozyme expression levels, localization or intracellular calcium concentrations. We further leveraged deep-learning-based rescoring to elevate site-level confidence. With this approach we mapped ~30,000 citrullination sites across ~5,500 of proteins, enabling direct comparison of substrate repertoire, sequence motif, and structural context among PAD isozymes, setting the stage for mechanistic dissection of how binding-pocket homology and peripheral residues dictate specificity. Distinct isozyme-specific preferences persisted in time-course experiments (10 min–16 h), indicating sequence context rather than temporal dynamics drives specificity. Mutation analysis of eleven PAD4 variants revealed Q346, G403S, R639, and H640 as key determinants distinguishing substrate recognition from that of PAD2. This work provides the most comprehensive PADs substrate atlas to date, defining isozyme-specific motifs and molecular determinants, and guiding development of selective inhibitors and probes to interrogate citrullination mechanisms in health and disease.