OBJECTIVE To evaluate the density of conjunctival goblet cells (GCs) in glaucomatous patients treated with preservative-free (PF) tafluprost, using laser scanning confocal microscopy (LSCM) and impression cytology (IC). METHODS Thirty glaucomatous patients (30 eyes) naive for therapy and thirty healthy subjects (30 eyes) were enrolled. Conjunctiva was examined by means of Heidelberg Retina Tomography/Rostock cornea module. Afterwards, the specimens for IC were obtained. Patients were randomized to PF-tafluprost (Group 1) or preserved latanoprost (Group 2) and controls to the vehicle of latanoprost (Group 3) or physiological buffered saline solution (Group 4). Both LSCM and IC were performed at baseline, and after the 1st and 6th months of therapy, GC density (GCD) (cells/mm(2) ) was the main outcome measurement. RESULTS Baseline. Mean GCD was 240.69 ± 25.43 and 232.65 ± 23.52, for LSCM, and 162.10 ± 23.44 and 164.71 ± 21.03 for IC in Group 1 and 2, respectively. GC density values were not significantly different in Group 3 and 4 (p > 0.05%). Month one. Mean GCD increased to 284.16 ± 43.88 and 230.62 ± 48.32 in Group 1 (p < 0.001) and to 297.86 ± 26.87 and 221.78 ± 43.02 in Group 2 (p < 0.05), measured with LSCM and IC, respectively. In Group 3, GCD decreased to 205.88 ± 25.04 and 139.54 ± 17.37 measured with LSCM and IC, respectively (p < 0.05). Month six. Mean GCD did not change in Group 1 (p > 0.05) whereas it decreased in Group 2 (p < 0.05), compared to month 1. In Group 3, GCD further decreased to 166.32 ± 22.31 and 120.76 ± 11.66, measured with LSCM and IC, respectively (p < 0.05); in Group 4, mean GCD did not change during the study period (p > 0.05). CONCLUSIONS Treatment with PF tafluprost was associated with an increase in conjunctival GCD in glaucomatous eyes naïve for therapy. Further studies are mandatory to verify this finding because its validation may have important consequences in the medical management of glaucoma.