In Endodontics, photosensitizers’ such as methylene blue and toluidine blue have been used in Photodynamic Therapy due to their positive results. However, they can stain the dentin from the root canal after Photodynamic Therapy (PDT). The present in vitro study aimed to evaluate different stain removal protocols from root canal after PDT using methylene blue (MB) dye. After mechanical preparation of the root canal of 40 uni-radicular human teeth, PDT was performed using 0,01% MB with parameters of 3 min of pre-irradiation and a diode laser irradiation emitting at 660 nm, 40 mW, 4 min, 9.6 J. After PDT, different protocols of MB removal were performed: Group 1 − control (0.9% saline solution); Group 2 − sodium hypochlorite (2.5% NaOCl); Group 3–17% ethylenediamine tetraacetic acid (EDTA); Group 4 − passive ultrasonic irrigation (PUI); The color of the dentin of the root canal was measured, before, immediately after the PDT and immediately after the cleaning using a spectrophotometer. The ΔE values found were statistically compared using the ANOVA and Tukey's tests (α=0.05). All the treatments lead to some cleaning of root canal after PDT, however, none of the treatments tested completely removed all staining caused by MB photosensitizer of the root canal. Among the treatments tested, PUI and Hypochlorite 2.5% promoted greater cleaning, with no statistically significant difference between them. In conclusion, within the protocols tested in the present study, no treatments were able to completely remove MB staining of the root canal after PDT.