The flavone C-glycosides in Viola yedoensis have been reported as the major active antiinflammatory, anti-cancer, and antioxidant components. However, due to the difficulty in obtaining standard compounds, little work has been done on the quantitative analysis of the multiple major flavone C-glycosides in V. yedoensis. In this study, a microwave-assisted extraction (MAE) and high performance liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry (HPLC-ESI-MS/MS) method in the selected reaction-monitoring (SRM) mode was developed and validated for the simultaneous separation and quantification of three major active flavone di-Cglycosides (apigenin 6,8-di-C-β-D-glucoside, apigenin 6-C-β-D-glucosyl-8-C-α-L-arabinoside, and apigenin 6,8-di-C-α-L-arabinoside) in V. yedoensis. The MAE conditions were optimized using an orthogonal design L9 (34). The extraction yields by MAE were the same or even higher than traditional extraction methods. All calibration curves showed good linearity (r2 > 0.9986) over their corresponding test ranges. The LOD ranged from 17 to 34 ng/mL and LOQ ranged from 34 to 68 ng/mL. The intra- and inter-day precisions (%RSD) were within 2.64% for the three analytes. The recovery of the assay was in the range of 97.67-98.93% with RSD values less than 2.54%. The developed HPLC-MS/MS method was used to quantitatively analyze the three di-C-glycosides in 14 batches of V. yedoensis samples. This newly developed HPLC-MS/MS method coupled with MAE is sensitive and accurate for simultaneous determination of multiple flavone C-glycosides in V. yedoensis.