A new and simple analytical method using HPLC with fluorescence detection was developed for the simultaneous determination of four lignans (phyllanthin, hypophyllanthin, phyltetralin and niranthin) in Phyllanthus niruri L. plant samples. Optimal separation was achieved with an isocratic mobile phase consisting of acetonitrile-water (55:45 v/v). The method recorded limits of detection (S/N=5) for phyllanthin at 0.61 ng/mL, hypophyllanthin at 6.02 ng/mL, phyltetralin at 0.61 ng/mL and niranthin at 1.22 ng/mL, being 80, 8, 80 and 40 times, respectively, lower when compared with those derived using HPLC-UV detection. The limits of quantification (S/N=12) were 4.88 ng/mL for phyllanthin and phyltetralin, 9.76 ng/mL for niranthin and 24.4 ng/mL for hypophyllanthin showing 40, 8 and 20 times, respectively, lower than those from the UV detection method. The within-day and between-day accuracy for the four lignans were between 98.1% and 102.9% while their precision values were below 2.2%. The mean recovery was between 92.5% and 110.1%. The method was then successfully applied for the quantification of lignans in P. niruri plant samples. The highest amount of lignans was found in the leaves followed by fruits, branches and stem, whilst the roots have the least amount of lignans.