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Interest has increased in the role of N‐acyl amino acids in a variety of disease states and as potential pharmacotherapies. Recently, N‐oleoyl glycine and N‐oleoyl alanine have shown promise in reducing the rewarding effects of drugs of abuse and alleviating withdrawal signs in rodent models. Previously published methods for the quantitation of these analytes by high‐performance liquid chromatography‐tandem mass spectrometry (HPLC‐MS/MS) in tissue were part of extensive lipidomic panels which may result in limited sensitivity and selectivity and also reported low recovery. Presented is a method for the extraction and HPLC‐MS/MS analysis of N‐oleoyl glycine and N‐oleoyl alanine. The bias and precision of the assay were determined to be within ± 20%. The method was shown to be reliable and robust, with over 90% recovery for the low‐level analytes. Increasing concentrations of N‐oleoyl glycine and N‐oleoyl alanine were quantitated in mouse brain and plasma following exogenous administration. This method was developed to serve to support studies investigating the pharmacokinetics and involvement of N‐oleoyl glycine and N‐oleoyl alanine in drug dependence and other diseases. [ABSTRACT FROM AUTHOR]