Abstract: Objective: To determine the content of 16 phenolic compounds in 24 batches of Prunella vulgaris by liquid chromatography- tandem mass spectrometry (LC-MS/MS) and to investigate the correlation between phenolic compounds and antioxidant activity by grey relational analysis (GRA) and partial least squares regression (PLSR) analysis. Methods: The sample was extracted with 80% methanol by the ultrasonic extraction. A method for the determination of 16 phenolic compounds in Prunella vulgaris was established by LC-MS/MS. The Waters ACQUITY UPLC BEH C₁₈ column (2.1 mm×100 mm, 1.7 μm) was selected as the stationary phase. The gradient elution was performed with the mobile phase of 0.1% formic acid-acetonitrile at the flow rate of 0.4 mL/min. The eluted compounds were detected in the negative ion mode with multiple reaction monitoring technology. The antioxidant activities were evaluated by DPPH free radical scavenging capacity, ABTS free radical scavenging capacity and ferric reducing antioxidant power. Results: There were significant linear relationship of 16 phenolic compounds in the range of their concentration with the correlation coefficient (r) greater than 0.99. The precision, repeatability, stability and recovery were good. In vitro antioxidant experiments showed that Prunella vulgaris had strong ability to scavenge ABTS, DPPH free radicals and ferric reduction. The results of GRA and PLSR indicated that rosmarinic acid, caffeic acid, rutin, hyperoside and isoquercitrin were the primary compounds contributing to the antioxidant capacity. Conclusion: A LC-MS/MS method for simultaneous determination of 16 phenolic compounds in Prunella vulgaris was established and the primary antioxidant active components were further investigated by correlation analysis, so as to provide scientific basis for quality control, development and utilization of Prunella vulgaris.