The dataset contains raw data files supporting the paper entitled "Development and validation of targeted metabolomics methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of 235 plasma metabolites". The data is organized into two separate dataset for both HILIC-MS/MS and RP-MS/MS methods. Each dataset contains 30 samples measured during the applicability test. Those samples were plasma from different sources, including QC porcine plasma (old and new batches), rat plasma, bovine plasma, chicken plasma, human plasma and NIST plasma. Two dilutions of 1:100 and 1:5 (v:v) were employed on each plasma sample except NIST samples that had one additional dilution of 1:400. For more information of sample type, dilution and method, please refer to the two Excel sheets, namely "HILIC_raw_files_list" and "RP_raw_files_list". The provider of plasma, work-up of plasma samples, procedure of applicability test and data processing can be found in "Materials and Methods" of the paper. The DOI for the paper will be linked here upon its publication.