Purpose <p>The present study aimed to develop a novel and green analytical method for the simultaneous analysis of Chloride, Sodium Citrate, Glycine, Niacinamide, D-Panthenol, and Pyridoxine in ORT LRG Liquid. The developed method was thoroughly validated by ICH Q2 (R1) guidelines to ensure its suitability for accurate and consistent quantitative analysis.</p> Methods <p>Analysis was performed using a RP-HPLC method with a C-18 column and a gradient elution technique. The method was optimized under suitable chromatographic conditions. The method was validated for system suitability, specificity, linearity, LOD, LOQ, accuracy, precision, and robustness. The method was also assessed using the greenness (AGREE, MoGAPI) and RAPI tools.</p> Results <p>The developed method was found to be in accordance with the ICH Q2 (R1) guidelines. The validation parameters remained within acceptable limits. The method demonstrated good suitability (tailing factor ˂ 2%, theoretical plates ˃ 2000), robustness, specificity without any notable interference, exhibited good linearity (R² ˃ 0.990), acceptable LOD, LOQ values, precision (RSD ˂ 2%), recovery rates (˃ 98%), AGREE (0.71), MoGAPI (79), and acceptable RAPI scores confirming the reliability and greenness of the developed method for Chloride, Sodium Citrate, Glycine, Niacinamide, D-Panthenol, and Pyridoxine.</p> Conclusion <p>The developed RP-HPLC method proved to be novel, reliable, precise, robust, and can be effectively applied in future research for the quantification of these compounds simultaneously in feed supplements, veterinary and pharmaceutical formulations containing a complex blend of electrolytes, buffering agents, amino acids, and vitamins.</p> Graphical Abstract <p></p>