A green, simple and sensitive HPLC method coupled with fluorescence detection was implemented for the quantitative determination of the anti-glaucoma drug tafluprost (TFL). Liquid chromatography was performed on HyperClone™ ODS (C18) column of dimensions; 150 × 4.6mm i.d. and 5μm particle size using a green eluent; ethanol:0.01M phosphate buffer (60:40v/v, pH4.5) delivered at 1mLmin-1. Fluorescence detection was accomplished at 220nm (excitation) and 292nm (emission). Bimatoprost (BIM) was used as an internal standard (I.S.). In this method, TFL was eluted after 6.70minutes. The method satisfied International Council for Harmonization (ICH) validation guidelines, as proved by good linearity (r= 0.9999, over the range 0.05-2μgmL-1), accuracy (recovery average 100.13 ± 1.27%), precision, robustness and specificity. The limit of detection and limit of quantification were found to be 0.016 and 0.048μgmL-1, respectively. The proposed method has been successfully applied for the estimation of TFL in eye drops and aqueous humor. For the first time, the approach was applied with acceptable results for the evaluation of the uniformity of TFL eye drops content. Furthermore, Green Analytical Procedure Index (GAPI) and analytical Eco-scale were used to prove that the proposed HPLC method is environmentally friendly.