A simple method for determining short-chain fatty acids (SCFAs) in rat and human feces was developed using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). A two-channel HPLC-ECD system was fabricated using an ion exclusion column and an electrochemical detector with a glassy carbon working electrode. Aqueous solutions of 0.1 mM HClO(4) and of ethanol containing 2-methyl-1,4-naphthoquinone served as a mobile phase and a quinone solution, respectively. Peak areas for lactic, acetic, propionic, butyric, isovaleric, and valeric acids at a detection potential of -0.9 V vs. an Ag/AgCl electrode showed a linear relationship with the acid amount in the range 0.1 to 40 nmol. Standard acids at 4 nmol were determined ten times with relative standard deviations (RSD) of less than 2.0%. The analytical results of healthy human feces were measured within 35 min. RSD (n = 5) in all SCFAs were less than 2.7%, and recoveries of SCFAs were more than 92%. The present method was characterized by reproducibility with the simple and rapid procedure without derivatization of analytes, and it has the potential for clinical and biomedical applications.