Both α‐ketoglutaric acid (KGA) and pyruvic acid (PYR) are important keto acids. Efficient co‐production of KGA and PYR has been achieved in our previous work, and could significantly decrease the cost of fermentation production. KGA and PYR have similar physical and chemical properties. Hence, finding a way to separate the two keto acids efficiently has become a key challenge. In this study, different chromatographic methods have been investigated, including ion‐exchange chromatography, aluminum oxide chromatography and silica gel chromatography. The results show that the two keto acids can be well separated with silica gel chromatography, whereas ion‐exchange chromatography and aluminum oxide chromatography could not separate them. Using the pretreated fermentation broth of Yarrowia lipolytica WSH‐Z06, the purity and yield of KGA/PYR reached 98.7%/99.1% and 86.7%/70.9%, respectively, with an optimized silica gel chromatography‐based procedure. This study provides an efficient method for separating KGA and PYR from fermentation broth, which might be applied on an industrial scale and significantly decrease the cost of biotechnological production of keto acids. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1370–1379, 2018