Acylcarnitines (ACs) play important roles in the fatty acid β-oxidation and are considered as diagnostic markers for many diseases. Accurate determination of ACs remains challenging due to their low abundance, high structure diversity, and limited availability of standard compounds. In this study, microwave-assisted Tmt-PP (p-[3,5-(dimethylamino)-2,4,6-triazine] benzene-1-sulfonyl piperazine) derivatization was utilized to facilitate the liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) determination of ACs. The result indicated that Tmt-PP labeling enables the prediction of the retention time and MS response of ACs and enhances their MS response up to 4 times. The introduction of the microwave during the derivatization procedure greatly improved the reaction efficiency, demonstrated by the shortened reaction time from 90 to 1 min. Furthermore, we applied a strategy named quantitative analysis of multi-components by a single marker (QAMS) for the assay of 26 ACs with only 5 AC standards, solving the standard availability issue to a large extent. The established workflow was applied to discover dysregulated ACs in xenograft colon cancer mice, and the quantification results were highly comparable with traditional methods where there were the corresponding standards for each AC. Our study demonstrated that chemical derivatization-based LC-MS/MS integrated with the QAMS strategy is robust for the identification and quantification of ACs and has great potential in targeted metabolomics study.