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Most common prostaglandin analogs, bimatoprost, latanoprost and travoprost, are licensed for the reduction of elevated intraocular pressure in patients with open angle glaucoma and ocular hypertension, but their non approved use as eyelash enhancers is becoming popular, especially in patients with eyelashes hypotrichosis. A fast and sensitive high performance liquid chromatography tandem mass spectrometry method was developed for the measurement of bimatoprost, latanoprost and travoprost in cosmetic serums freely web-sold to increase eyelash length, thickness and darkness. The analytes and the internal standard (reserpine) were separated by reversed phase chromatography with 5 mM ammonium acetate with 0.02% formic acid (mobile phase A) and 5 mM ammonium acetate in acetonitrile/water (95/5; v/v) with 0.02% formic acid (mobile phase B) by gradient elution and detected with tandem mass spectrometry operated in multiple reaction monitoring mode. Linearity between 1 and 500 μg/g shows good correlation coefficients (r2 = 0.99) for all substances. Analytical recovery of analytes under investigation were always higher than 90% and intra-assay and inter-assay precision and accuracy always better than 11%. This method was successfully applied to analyze cosmetic serums freely sold on the Internet websites.