Context: In regenerative endodontic techniques, a blood clot acts as a natural scaffold for revitalizing dental pulp tissue. Although 17% ethylene diamine tetra acetic acid (EDTA) releases growth factors from the dentin matrix and induces odontoblast differentiation, it has anticoagulant property. Aims: The aim of the study is to evaluate the ability of alternatives (0.2% chitosan and papain) in fibrin formation when used as root canal irrigants during regenerative endodontic procedures in teeth with open apices. Also investigated whether 0.2% chitosan and papain solutions can be used as alternatives to 17% EDTA-normal saline solution (EDTA-NSS) combination, as root canal irrigants during regenerative endodontic procedures in teeth with open apices. Subjects and Methods: Twenty-five human freshly extracted single-rooted mandibular premolars were taken. They were then divided into five groups and irrigated accordingly. Human donor blood samples were collected and placed in the specimens and fibrin density was evaluated using scanning electron microscopy. Comparison of fibers per 10 μm among 5 experimental groups at each level was performed using one-way analysis of variance test. Results: Results revealed that the specimens irrigated with EDTA-NSS followed by papain had an increased fibrin density when compared with chitosan. Conclusions: All the root canal irrigants that were used in this study as part of treatment protocol for open apex exhibited positive results. © 2022 Elsevier B.V., All rights reserved.