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A solid-phase extraction (SPE) process, followed by an HPLC-DAD method, was developed and validated to quantify short-chain fatty acids (SCFAs) and applied to analyze chicken feces samples. This study aimed to report the use of the STRATA^TM-X-A 96 Well Plate SPE cartridge method as the first step in quantifying SCFAs. A stationary reverse-phase Luna Omega C18 column kept at 40 °C was used, with a gradient elution mobile phase (H₂SO₄ and Acetonitrile, 98:2), a flow rate of 1.2 mL/min, and detection at 210 nm. A mixture of acetic, propionic, and butyric acid was used as thestandard. The method showed a linear relationship, with a coefficient of determination of R² = 0.9987, R² = 0.9985, and R² = 0.9966 for acetic, propionic, and butyric acid, respectively. Concerning sensitivity, an LOD and LOQ of 0.14, 0.14, 0.14 mg/mL and 0.44, 0.45, 0.43 mg/mL were obtained for acetic, propionic, and butyric acid, respectively. According to the sample analysis, the accuracy was 76.05, 95.60, and 81.56% for acetic, propionic, and butyric acid, respectively. The developed method is simple, fast, linear, sensitive, accurate, precise, and robust for the quantification of SCFAs. This could serve as an alternative to conventional methodologies for the determination of these critical components in the intestinal health of chicken feces.