This project focuses on developing highly specific monoclonal antibodies for detecting citrullinated proteins while minimizing cross-reactivity with homocitrullination. Citrullination, a post-translational modification catalyzed by PAD enzymes, plays key roles in immunity, inflammation, and disease. However, current detection methods lack sensitivity and specificity, limiting their utility. To address this, we employed a motif-based approach using PAD sequence preferences to generate monoclonal antibodies. Rats were immunized with a pool of over 490,000 citrullinated peptides representative of human tissue proteomes, and two antibody clones could be established and validated using ELISA. For further testing, citrullinated and homocitrullinated samples were generated in vitro, and the presence and content of the respective modification confirmed using mass spectrometry. These samples, as well as ionomycin-activated human neutrophils, were used to evaluate antibody performance in western blotting, where they demonstrated high sensitivity, specificity, and quantitative detection capabilities. These novel antibodies provide valuable tools for studying citrullination dynamics and have promising applications in biomarker discovery and disease diagnostics.