Abstract: A sensitive and specific HPLC-MS/MS method was developed for the quantitative determination of olopatadine in human plasma. Plasma samples were prepared by Tomtec automatic SPE device and separated on an Alltima C₁₈ column with methanol∶0.05% formic acid＝50∶50 (v/v) as the mobile phase. Detection was performed on a triple-quadrupole tandem mass spectrometer using positive electrospray ionization (ESI), and multiple reaction monitoring (MRM) was applied. The results showed the lowest limit of quantitation of olopatadine was 0.1 ng/mL, and the linear range was 0.1~100 ng/mL. Accuracies and precisions of all were within ±15%. This method proved to be specific, sensitive and accurately, which can be applied to determine olopatadine in human plasma.