The removal or modification of smear layers that cover the dentin is critical to allow the penetration of adhesive molecules and to ensure a strong bond between resin and dentin. Aiming to establish a model for clinically-relevant dentin-bond testing, we evaluated the effects of smear layers created by abrasives having similar coarseness (180-grit SiC paper; fine-grit diamond bur) and application modes (single application; double application) on the microtensile bond strengths (µTBS) of two currently available universal adhesives (G-Premio Bond; Scotchbond Universal Adhesive) and a two-step self-etch adhesive (Clearfil Megabond 2). Sixty extracted human third molars were used for the μTBS test. Data were analyzed by three-way ANOVA and Tukey's test (α = 0.05). Fracture modes were determined using stereomicroscopy. An additional 24 third molars were prepared for observation of the resin-dentin interface by TEM and adhesive-smear layer interaction by SEM. μTBS was significantly affected by the adhesives and their application modes (p < 0.001), implying that the double application of universal adhesives should be recommended to improve their performance. The effect of smear layers was not significant (p > 0.05), indicating that 180-grit SiC papers could be used to prepare dentin as a substitute for fine-grit diamond burs for dentin-bond testing in laboratory settings.