Objective: The aim of this study was to evaluate the permeability of five desensitizing agents using computerized fluid filtration (CFF) test method. Materials and Methods: Sixty dentin discs of 500 200-mm-thick were prepared from middle dentin of bovine incisors without exposed the pulp and then randomly divided into five groups (n = 12). The permeability of the discs was measured using the CFF test method before and after application of the following desensitizers: Admira Protect (Voco, Cuxhaven, Germany), Seal and Protect (Dentsply, Konstanz, Germany), Sensi Kill (DFL, Brazil), Systemp Desensitizer (Ivoclar Vivadent, Liechtenstein), BisBlock (Bisco, USA). Fluid movement measurements were made at 2-min intervals for 8 min, and a mean of the values obtained was calculated for each specimen. The results were analyzed using Kruskal-Wallis test and Wilcoxon signed ranks tests with a significance threshold of P < 0.05. Results: There were no significant differences in permeability among desensitizing agents (P > 0.05); however dentin permeability was reduced in all groups (P < 0.05). Conclusion: The in vitro fluid conductance of dentin discs were reduced by treating with these five desensitizing agents.