Introduction: The stabilization of dentinal collagen fibers against enzymatic degradation by the use of biocompatible cross-linker agents is of clinical importance for effective dentin bonding to surpass the test of time. Objective: The present study aims to evaluate and compare the effect of the application of two versions of a desensitizer solution to sound coronal dentin, on the microtensile bond strength (μTBS) of the resin-sound coronal dentin using 4th and 6th generation dentin bonding systems. Materials and Methods: Extracted human third molars were collected from an unidentified bank of teeth followed by IRB approval. A flat surface of all 12 teeth was prepared utilizing a water-cooled high-speed diamond disc, leaving an entire hard sound dentinal area for testing. Subsequently, according to the assigned group, specimens followed specific manufacturer’s instructions for application of dentin bonding systems: specimens were subdivided into 6 groups (n=20). Group 1 (G1) First positive control group. Specimens received an application of a 4th generation dentin bonding system (DBS). Group 2 (G2) Second positive control group. Specimens received an application of a 6th generation DBS. Group 3 (G3) Specimens were exposed to Gluma Desensitizer agent, blot-dried and followed by application of a 4th generation DBS. Group 4 (G4) Specimens were exposed to Gluma Desensitizer agent, blot-dried and followed by application of a 6th generation DBS. Group 5 (G5) Specimens were exposed to Gluma Desensitizer PowerGel agent, blot-dried and followed by application of a 4th generation DBS. Group 6 (G6) Specimens were be exposed to Gluma Desensitizer PowerGel agent, blot-dried and received an application of a 6th generation DBS. After application of the adhesive systems, all specimens were restored using a microhybrid resin composite. The root portion was sectioned 1mm below the CEJ, and discarded. All specimens were thermocycled at 5-55 Cº for 7000 cycles on distilled water. Then each restored tooth was sectioned perpendicular to the bonding interface into 1mm x 1mm x 8mm beams with a slow speed diamond wafering blade under thorough irrigation. Then specimens were subjected to μTBS testing at a crosshead speed of 1mm/min. Subsequently; specimens were subjected to fracture analysis and SEM evaluation of the different failure’s mode of the involved surfaces. Statistical analysis was performed by usingone- way ANOVA, two-way ANOVA and Fisher’s PLSD test (p<0.05). Results: For the first aim of the study and after obtaining the μTBS in MPa: Group G1: 15.50 ± 6.28, Group G2: 13.06 ± 11.53, Group G3: 19.20 ± 9.43, Group G4: 12.76 ± 4.61, Group G5: 14.38 ± 5.95, Group G6: 18.54 ± 9.49. Statistical analysis showed that there is no significant influence of variables on the μTBS (Welch ANOVA [F (5,114) =2.21, p=0.057]). Treatment with Gluma desensitizing agent and Gluma desensitizing PowerGel has no significant influence on the bond strength. For the second aim of the study and to analyze group differences for type of fracture data was first recoded into two groups: (1) Adhesive failure and (2) Cohesive failure. Group differences were analyzed by type of fracture using a Fisher’s exact test. No difference was found between the groups by type offracture (5, N = 120) = 8.62, p = 0.090 Conclusion: Within the limitations of this in vitro study it can be concluded that Gluma desensitizing agent and Gluma desensitizing PowerGel did not significantly affect the μTBS of both 4th and 6th generation bonding system using extracted human teeth.