Simultaneous detection of various o-phthalaldehyde (OPA)-labeled amino acids (AAs) in food samples was reported based on CE separation. Ionic liquid was used for the first time for CE analysis of AAs with in-capillary derivatization. Several other additives, including SDS, /-CD, and ACN, as well as key parameters for CE separation (buffer pH value, separation voltage), were also investigated. Our results show that the multiple additive strategy exhibits good stable and repeatable character for CE analysis of OPA-labeled AAs, for either in-capillary derivatization or CE separation, and allows simultaneous quantification of different OPA-labeled AAs in a large concentration range of 50 M to 3.0 mM with LOD down to 10 M. Seventeen OPA-labeled AAs, except for two pairs of AAs (His/Gln and Phe/Leu), which were separated with resolutions of 1.1 and 1.2, respectively, were baseline separated and identified within 23 min using the present multiple additive strategy. The method was successfully applied for simultaneous analysis of AAs in seven beer samples and as many as eleven trace-amount AAs were detected and quantified, indicating the valuable potential application of the present method for food analysis.