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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 …