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Piper is a genus that is recently valued for the treatment of central nervous system diseases. The major constituents, amides and lignans, are responsible for the antinociceptive and antidepressant activities. This study developed a RP-HPLC-UV method for the simultaneous determination of eight amides and two lignans in twelve different species of Piper. HPLC separation was accomplished on a C18 analytical column (5 μm, 250 mm × 4.6 mm, i.d.) with a gradient mobile phase consisting of acetonitrile and water at a flow rate of 1.0 ml/min. All the calibration curves showed good linear correlation coefficients (r > 0.9997) over the test ranges. The relative standard deviation of the current method was less than 2.90% for intra- and inter-day assays and the average recoveries were between 98.25% and 103.08%. The HPLC method established is appropriate for quality control purposes and allows for the differentiation of Piper species.