Purpose <p>This study aims to develop and validate a novel reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of lysozyme and dequalinium, two compounds with antimicrobial and anti-inflammatory properties. The research seeks to address the need for a reliable analytical tool for combination therapies targeting infections and inflammation, the study design is summarized in the Graphical Abstract.</p> Methods <p>The RP-HPLC method was optimized using a Zorbax 300 SB-C18 column at 60&#xa0;°C, with a gradient mobile phase of trifluoroacetic acid and acetonitrile. The method was validated for linearity, detection limits, accuracy, precision, and robustness following ICH guidelines. Analytical performance was assessed in pharmaceutical formulations containing lysozyme and dequalinium.</p> Results <p>The method demonstrated excellent linearity (r2 &gt; 0.999) in the concentration range of 3–50&#xa0;µg/mL for both compounds. Detection limits were 2.60&#xa0;µg/mL for lysozyme and 2.06&#xa0;µg/mL for dequalinium. Recovery rates were close to 100%, indicating high accuracy and precision. The method also displayed robust performance with minimal variation in results.</p> Conclusions <p>The developed RP-HPLC method is efficient, reliable, and suitable for the simultaneous quantification of lysozyme and dequalinium in pharmaceutical formulations. It offers a promising analytical tool for quality control in combination therapies and demonstrates exceptional sustainability, as indicated by high greenness, blueness, and whiteness scores.</p> Graphical Abstract <p></p>