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Volatile fatty acids (VFAs) are the important intermediates indicating the stability and performance of fermentation process. This study developed the spectrophotometric method for determining high-range VFA concentration in mixed-acid fermentation samples. The performance was compared with the gas chromatography (GC) technique. The calibration curves of the modified method showed linearity over a wide and high concentration range of 250–5000 mg/L for individual C2–C6 VFAs in both linear and branched chains. In order to evaluate the modified method for VFA determination in complex fermentation matrices, fermentation samples produced from acidogenic fermentation of plant materials were spiked with acetic (500–1500 mg/L) and butyric acids (1000 mg/L). The accuracy and precision of the modified method for VFA determination were in the range of 94.68–106.50% and 2.35–9.26%, respectively, comparable to the GC method (94.42–99.13% and 0.17–1.93%). The developed method was applicable to measuring all C2–C6 compounds and VFA concentrations in the fermentation samples and had an acceptable accuracy and precision. The proposed method is analytically reliable and offers significant advantages in the rapid determination of VFAs in mixed acid fermentation of organic residues.