Background: The establishment of appropriate working length is one of the most critical steps in endodontic therapy. There is alteration in accuracy of apex locators in presence of irrigants, pulp/blood and open apices. Hence their accuracy in these conditions needs to be evaluated. Aim: The aim of this in vitro study was to determine the accuracy in measuring the working length by using CanalPro, Apex ID and Root ZX mini Apex locators in different simulated clinical conditions. Materials and Methods: 60 freshly extracted single-rooted teeth were equally assigned to three groups according to simulated clinical conditions: Group 1; Presence of irrigant, Group 2; open apex, Group 3; presence of blood & pulp. The working length was determined with all the apex locators and was then compared with actual working length of the tooth. The difference between the length measured by electronic apex locator (EAL) and actual length (AL) was tabulated and statistically analyzed using one way ANOVA and Post hoc Bonferroni test. Results: The accuracy in working length measurement by Root ZX mini was 100%, 60% and 80%; Apex ID was 100%, 80% and 90% and CanalPro was 100%, 90% and 90% in group 1, group 2 and group 3 respectively within ± 0.5 mm of AL. ANOVA revealed that comparison of the mean difference between length measured by all EAL’s and AL was statistically insignificant in group I. In group II and III, CanalPro showed the least difference between AL and length measured by it though it was statistically insignificant as compared to Apex ID. Root ZX showed the maximum difference between actual length and length measured by it. Conclusion: CanalPro showed highest accuracy in all conditions with accepted accuracy percentage above 90%.