Objective: The aim of this study was to evaluate the effect of different irrigation methods on sodium hypochlorite (NaOCl)/debris extrusion and dentinal tubule penetration of 2.5% NaOCl in the presence of an apically separated instrument. Materials and methods: Sixty root canals of freshly extracted mandibular single-rooted premolars were chemomechanically prepared up to ProTaper F2. Manual needle irrigation (30-gauge/side-vented) with 2.5% NaOCl was performed between files and a smear layer was removed with 17% ethylenediaminetetraacetic acid. Three mm of notched ProTaper F3 files were separated in the apical third of the roots. Samples were then randomly divided according to the final irrigation systems as follows: EndoActivator, EndoVac, manual needle irrigation, passive ultrasonic activation, and RinsEndo. Samples were mounted to Eppendorf tubes and root canals were irrigated with 3-mL Rhodamine B-labelled 2.5% NaOCl, which was applied using one of the above-mentioned methods. The extruded NaOCl/debris weight was calculated by extracting pre-irrigation weights of tubes from post-irrigation ones. Samples embedded in acrylic resin were sectioned transversely in 1-mm thicknesses at apical 1- and 3-mm levels. NaOCl's penetration depth and percentage into the dentinal tubules were evaluated with the aid of a confocal laser scanning microscope. Data were analysed statistically with Kruskal-Wallis and post hoc Siegel-Castellan tests (p < .05). Results: EndoVac significantly decreased the extruded NaOCl/debris compared to RinsEndo, passive ultrasonic activation, and EndoActivator in the presence of a separated instrument (p < .05). The penetrability of NaOCl significantly increased with the use of RinsEndo and EndoVac compared to the remaining groups (p < .05). Conclusion: In the presence of an irretrievable separated instrument, it could be suggested that devices with apical negative pressure such as EndoVac may improve the penetrability of irrigation solutions to the apical part of the root while preserving periapical tissues from NaOCl/debris extrusion.