Objective:To evaluate the effect of final rinsing solution on smear layer removal through penetrability of the root canal sealer.Materials and Methods:Each of the 30 teeth selected was decoronated and the root length standardized at 16 mm measured from the apex of the tooth to the cementoenamel junction (CEJ). A size 10-k file was used and working length was determined by subtracting 1mm from the measured length so that the length of the sample was 15mm. A combination of step back and crown down technique was done and apical portion was prepared till H-file no. 30. 3% NaOCl was used during cleaning and shaping of the canals. Finally the teeth were divided into three groups of 10 teeth each. Group-I was irrigated with 17% EDTA, Group-II with TUBLICID plus, and Group-III with Biopure MTAD®. ISO 30 size gutta-percha points were selected as master cones. The Acroseal sealer was mixed with approximated concentration 0.1% fluorescent rhodamine B isothiocyanate dye. Obturation was done and after 48 hours, the roots were sectioned and subjected to confocal laser scanning microscopy.Results:Comparisons among the three groups were done by two-way analysis of variance (ANOVA) and Newman-Keuls multiple post-hoc procedures. The two-way ANOVA showed statistically highly significant results. The maximum depth of sealer penetration was observed in Group II followed by Group III and Group I.Conclusion:The use of -Tubulicid plus as a final irrigant provided better smear layer removal than Biopure a mixture of doxicycline, citric acid and a detergent (Tween 80) (MTAD®) and 17% EDTA.