Introduction: The aim of this study was to to evaluate and compare the efficacy of manual dynamic activation, passive ultrasonic irrigation and laser in removal of triple antibiotic paste (TAP) from root canal walls. Methods: Thirty-six extracted human mandibular premolars were prepared using ProTaper rotary files up to size F3. The roots were divided longitudinally and a standardized groove was prepared in the apical part of one segment. The root halves were assembled after inserting TAP into the grooves. According to the irrigation protocol used, the specimens were randomly divided into three experimental groups: manual dynamic activation, passive ultrasonic irrigation (PUI) and laser activated irrigation using with 5.25% NaOCl and 17% EDTA. The root segments were separated, and a four-grade grading system was used to determine the remaining TAP under a stereomicroscope at 10x magnification. The data were evaluated statistically using Student t test and ANOVA test with a 95% confidence level (P = 0.05). Results: There were significant differences amongst the groups (P < 0.001). Passive ultrasonic irrigation (PUI) and laser assisted irrigation removed significantly more TAP than manual dynamic activation. However, no significant difference was seen among the passive ultrasonic irrigation and laser assisted irrigation. Conclusions:PUI was most successful at removing TAP from root canal artificial grooves. However, TAP could not be totally eliminated from root canals.