Abstract: To evaluate an effective cleaning protocol for endodontic instruments. Total 180 new endodontic k-files (sizes 15, 25 and 40) were contaminated by preparing canals of extracted teeth. The practices used different cleaning protocols for the instruments. These protocols involved four components the use of sponges soaked with chlorhexidine, brushing, pre-soaking in MICRO 10 ENZYME and ultrasonication. After cleaning, the files were immersed in Van Gieson’s solution and examined under magnification for stained debris. The effectiveness of cleaning sequences was tested on the instruments. The mean percentage of highest contamination score was 35% in sponge soaked with chlorhexidine gluconate group, 15% in the brushing only group, 5% in MICRO 10 ENZYME soak plus brushing group, 0% in MICRO 10 ENZYME soak and brushing and ultrasonication group and 4% in ultrasonicatin only group. There were no clean instruments in these groups except group 4. There was a statistically significant difference between the full cleaning method (group 4) and other groups (p<0.05). Statistically, size of instruments did not significance influence on debris removal. The best method for the efficient debris removal was the one that included mechanical, chemical and ultrasonic cleaning of instruments.