The aim of this study was to evaluate in vitro dentine permeability evaluation of 2-Hydroxyethyl methacrylate (HEMA)-based desensitizing products using split-chamber model following in vivo application in the dogs for three experimental time periods of 1 week, 1 month and 3 months. Buccal enamel of upper and lower canines of nine young dogs was removed and flat dentinal surface was obtained using a water-cooled diamond bur. The dentinal surface divided into four quarters, three experimental and a control. Health-Dent and Gluma Desensitizing agent and Single bond were applied to respective quarters of one tooth, according to manufacturers' instructions. The last quarters were left as control without any applications. For the each experimental period, 10 dentine samples from each group including control were used to measure the hydraulic conductance values. The dentine discs were placed pulp-side down in a split-chamber device in which the plastic spacers containing the rubber 0 rings have a surface area of 1 mm(2) and permeability was measured by fluid filtration. The data were expressed as hydraulic conductance (Lp). Differences in dentine permeability obtained for the desensitizing agents against the control were tested for statistical significance using Kruskal-Wallis one-way ANOVA and Bonferroni-adjusted Mann-Whitney U-tests. Differences in permeability in three time periods for each desensitizing agent were analysed using Friedman's anova and Wilcoxon signed rank tests. All desensitizing applications caused decreased Lp values compared with the control Lp value at the end of 1 week. In the 1-month samples, between the Lp values of Single bond and control, no statistical difference was found (P > 0.05). At the end of the 3-month period, the Gluma Desensitizing agent had the lowest the Lp value (P < 0.05). The result of this study is that the topical application of desensitizing agents led to decrease in dentine permeability in the dog model. The Gluma Desensitizing agent provide the more lasting tubule-occluding effect than the other material tested in this model.