A lyophilization-supercritical fluid extraction coupled with supercritical fluid chromatography-MS/MS online method was developed for the determination of lipid mediators in breast cancer cells. Supercritical fluid extraction was applied to the cell samples for the first time due to the use of lyophilization. The conditions of supercritical fluid extraction and supercritical fluid chromatography-MS/MS were investigated systematically. Under the optimized conditions, all the calibration curves for the lipid mediators showed good linearity (R  > 0.99). The limits of detection and the limits of quantification were in the range of 0.190-5.36 pg and 0.560-16.2 pg, respectively. The recoveries were in the range of 70.3%-125%. The relative standard deviations of the precision ranged from 1.49%-18.7% and the accuracies were higher than 84%. Compared with liquid-liquid extraction coupled with LC-MS/MS method, the present approach reduced the manual labor and obtained higher sensitivity as well as higher extraction recoveries for all 15 lipid mediators. Finally, the online method was applied to the quantification of lipid mediators in breast cancer cells and normal mammary epithelial cells. On the basis of the results, this lyophilization-supercritical fluid extraction online coupled with supercritical fluid chromatography-MS/MS method showed great promise in the analysis of lipid mediators in complex biological samples. This article is protected by copyright. All rights reserved.