Analysis of fatty acids takes considerable attraction in metabonomics researches because the metabolism of fatty acid is closely related to energy metabolism, signal transduction and some disease occurrence.Although many separation and quantitative methods for fatty acids have been developed, most of them remain time consuming, especially, for the large-scale analysis of clinic urine samples.In this work, a rapid and sensitive method for analysis of free fatty acids from plasma or urine samples by ultra performance liquid chromatography-quadrupole time of flight mass spectrometry was developed.In this way, 19 fatty acids including two pair of hard-resolved isomers were separated well in 13 minutes as shown in Fig.1, and the method provides good accuracy, reproducibility and wide linear range.This method is applicable for high throughput analysis of free fatty acids analysis in plasma or urine samples.