Reproducible, skilfully conducted and unbiased laboratory studies provide new knowledge, which can inform clinical research and eventually translate into better patient care. To help researchers improve the quality and reproducibility of their research prior to a publication peer‐review, this paper describes the process that was followed during the development of the Preferred Reporting Items for Laboratory studies in Endodontology (PRILE) 2021 guidelines and which used a well‐documented consensus‐based methodology. A steering committee was created with eight individuals (PM, RO, OP, IR, JS, EP, JJ and SP), plus the project leaders (PD, VN). The steering committee prepared an initial checklist by combining and adapting items from the modified Consolidated Statement of Reporting Trials checklist for reporting in vitro studies of dental materials and the Clinical and Laboratory Images in Publications principles as well as adding several new items. The steering committee then formed a PRILE Delphi Group (PDG) and PRILE Online Meeting Group (POMG) to provide expert advice and feedback on the initial draft checklist and flowchart. The members of the PDG participated in an online Delphi process to achieve consensus on the items within the PRILE 2021 checklist and the accompanying flowchart for clarity and suitability. The PRILE checklist and flowchart developed by the online Delphi process were discussed further by the POMG. This online meeting was conducted on 12 February 2021 via the Zoom platform. Following this meeting, the steering committee developed a final version of the PRILE 2021 guidelines and flowchart, which was piloted by several authors when writing up a laboratory study for publication. Authors are encouraged to use the PRILE 2021 guidelines and flowchart to improve the clarity, completeness and quality of reports describing laboratory studies in Endodontology. The PRILE 2021 checklist and flowchart are freely available and downloadable from the Preferred Reporting Items for study Designs in Endodontology website (http://pride‐endodonticguidelines.org/prile/).