Abstract: Data independent acquisition (DIA) mode, is a novel multi-stage mass spectrometry (MS) acquisition technology and it can achieve rapid detection of full MS and all product ion spectra in a single liquid chromatography coupled with mass spectrometry (LC/MS) run. Sequential Windowed Acquisition of all Theoretical fragment ions (SWATH) is one of DIA mode. The scanning range of SWATH acquisition is divided into multiple continuous intervals which contains a certain width (usually 25 u), and all ions in the interval are fragmented and quickly scanned in order to acquire all MS and MS/MS information. On one hand, it can provide relatively simple MS/MS spectra of ions due to its relatively narrow m/z isolation window when comparing with other DIA mode, so it is widely used in untargeted metabonomics analysis. On the other hand, it can exhibit comparative sensitivity and quantitative accuracy when comparing with multiple reaction monitoring (MRM) and can be used for quantitative analysis. Thus, SWATH acquisition can show the advantage of high sensitivity, high coverage and high scanning speed. In this study, a simultaneous qualitative and quantitative analysis of multiple metabolites in the plasma was developed based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) with SWATH acquisition. 93 metabolites in the plasma can be successfully identified by home-made metabolites database searching which consists of metabolites related to cancers based on our previous metabonomics studies, thus achieving untargeted analysis. Quantification method was further developed for identified metabolites and methodology was examined. The analysis result shows that calibration curves were linear (the linearity of 95% metabolites more than 0.99) with limits of detection from 1.25 to 12 000 μg/L. In order to ensure the reliability of method, accuracy and stability of the method were further investigated, and 86 of metabolites in the plasma can achieve the quantification requirements in biological samples; Intra-day relative standard deviation (RSD) and inter-day RSD are both lower than 20%, and stability for 96 hours at 4 ℃ is from 0.62% to 19.35%. It’s worth noting that for metabolites with low content in the plasma or serious matrix interference, quantitation by its product ion can exhibit better linearity, wider dynamic ranges and lower limit of quantitation. The established method provides a reliable and rapid method for identifying and quantifying multiple metabolites simultaneously in the plasma with high coverage and high sensitivity, and it is also applicative in the analysis of other biological samples.