The use of enzymatic chemical agents are papain-based materials used in the selective removal of decayed dentin tissue, assisting in conservative techniques and reducing the chances of accidental pulp exposure. A research protocol was subjected to and approved by an Ethics Committee. Using a reporting guide for laboratory studies (CRIS). Healthy human teeth comprised the produced dentin discs subjected to polishing and washing in an ultrasonic bath. Next, the discs received material application according to the experimental groups: water-soluble gel for two minutes, 37% phosphoric acid for 15 seconds in dentin and 30 seconds in enamel, Papacárie Duo (PD) for 30 seconds and two minutes, and Brix 3000 (BX) for two minutes and 30 seconds. The measurement of material pH used solutions at concentrations of 0.1 ml and 2.7 ml prepared for each enzymatic agent. Then, a bench pH meter (n=10) and pH indicator strips determined pH values. The discs underwent the Knoop hardness test (n=10). The sample calculation was performed using the GPower software with α = 0.05, effect 0.63 and power of 95%. Descriptive statistical analysis was carried out for pH, one way ANOVA supplemented by Tukey for knoop hardness and Spearman correlation for pH measurement techniques. The enamel hardness findings indicated that, after material application, ECAs do not statistically differ from water-soluble gel (p<0.05). The dentinal hardness analysis presented a statistical difference in phosphoric acid from the other groups (p<0.05). In the pH test, BX values were lower (4.37 ± 0.01) than PD (4.85 ± 0.06). The groups statistically differed (p<0.05). ECAs for removing decayed dentin tissue did not significantly alter the hardness of enamel and dentin, removal of the smear layer is time-dependent and presents acidity. Key words:Dental caries, Hardness, Dental materials, Papain.