To establish a method for qualification and quantification of gallic acid in Hedyotis diffusa Willd. and to investigate its proapoptotic mechanism on lung cancer cells. Gallic acid content was determined by HPLC under the following conditions: stationary phase: Diamonsil C18 column; mobile phase: 0.2% methanol acetonitrile-0.1% phosphoric acid: 0.1% triethylamine aqueous solution (3:97); detection wavelength: 216 nm; and column temperature: 40°C. Apoptosis was detected with a flow cytometer by observing the apoptotic cell morphology, and livin, survivin protein expressions were detected by Western blot. Injection amount of gallic acid was in a good linearity with the peak area at a range of 0.02-0.41 μg, r=0.9997. Average recovery was 99.55%, RSD=2.25%. Hedyotis diffusa Willd. extract had a dose-dependent proapoptotic effect on NCI-H460 cells and suppressed the expressions of surviving and livin proteins in NCI-H460 cells. Quantitative determination of Hedyotis diffusa Willd. of different origins proves that the method is sensitive, accurate, reliable and reproducible. Induction of apoptosis in NCI-H460 lung cancer cells is achieved primarily by inhibiting the NCI-H460 cells from expressing survivin and livin, which provides a theoretical basis for use of Hedyotis diffusa Willd. extract for lung cancer treatment.