Objective To establish determination method of formic acid, lactic acid, acetic acid and succinic acid in dental plaque with high performance liquid chromatography(HPLC). Methods After the samples were cen - trifuged, 2 μL supernatant was transferred to a 1 mL centrifuge tube and diluted in water, then was determined with HPLC. The mixture of phosphate buffer and methanol(97∶3) as mobile phase throughout the experiment. The determination of organic acid was performed on Phenomenex C18 column and at their maximum absorption wave. Results The linear ranges of formic acid, lactic acid, acetic acid and succinic acid were 0.110 ~500, 0.049~500,0.047~ 500,0.084~500 μg/mL. The detection limits were 0.110,0.049,0.047,0.084 μg/mL. The relative standard derivation were 9.5%,7.9%,4.3%, 4.2%. The average recoveries of samples were 82%~112%,82%~102.5%,90%~ 115%, 80%~110%. Conclusion The method was simple, quick and adapt for analysis of organic acid in dental plaque.