Abstract In ruminant animals, volatile fatty acids (VFA) or short-chain fatty acids (SCFAs) are derived from the protein and carbohydrate fermentation by rumen microorganism. Hence, the VFA determination in rumen fluid allows the evaluation of the nutritional quality of a diet, as well as its potential impact on the chemical composition of ruminant milk and meat. Thus, we developed a protocol to extract, identify, and quantify acetic, propionic, butyric, valeric, and caproic acids in ruminal fluid samples using RP-HPLC-DAD. Despite literature findings had shown that the most suitable column for VFA chromatographic separation under HPLC-DAD is an ion-exchange column, our protocol showed that a C18 column also allows an efficient VFA separation of the aforementioned acid, except for butyric and iso-butyric acids. This condition may constitute a limitation of the Hypersil GOLD C18 column use for VFA determination. However, considering that the concentration of iso-butyric acid is significantly lower than that of butyric acid, a good estimation of butyric acid can be obtained.