A rapid and reliable LC-MS/MS method was developed for the quantitation of major components in Folium Artemisiae Argyi (mugwort), a widely used traditional Chinese herbal medicine. A total of 5 phenolic acids and 17 flavonoids were separated and simultaneously determined by using a Shiseido C column (150 × 3.0 mm, 3 μm) and gradient elution of acetonitrile-aqueous formic acid (100:0.1, v/v) at a 0.5 mL min flow rate, via multiple reaction monitor (MRM) in polarity switching mode. The quantitative method was validated in terms of sensitivity, linearity, precision, accuracy and stability, which proved to be sensitive, accurate and reproducible. Then 65 samples collected from different areas were selected for component analysis by LC-MS/MS and assessment of antioxidant activity using DPPH, ABTS, FRAP, O and OH scavenging assays. Grey relational analysis and partial least square regression were used to evaluate the relevance between chemicals and bioactivities, and the results indicated chlorogenic acid, isochlorogenic acid B, A, C, eriodictyol, jaceosidin and eupatilin made the key contribution to antioxidant activity. The present study combines chemical analysis and bioassay to identify bioactive markers, which possesses potential value for the activity-oriented quality control of mugwort.