The aim of this systematic review was to perform a qualitative synthesis of in vitro studies which evaluate and compare the penetration of calcium silicate-based sealers into dentinal tubules. A systematic advanced search was performed in Scopus, Embase, Medline (via PubMed), Web of Science, and Cochrane databases on the 1 December 2022. In vitro studies that compared the tubular penetration of at least two calcium silicate-based sealers in extracted human teeth were eligible. PRILE 2021 guidelines were used for the assessment of the risk of bias included studies. The search identified a total of 680 preliminary records, among which 12 studies were eligible for review. The most used methodology to evaluate sealer penetration was the use of a fluorochrome together with the tested sealers and the analysis of their penetration under confocal laser microscopy. Regarding the results of the included studies, calcium silicate-based sealers exhibit a favorable dentinal tubule penetration. Tubular penetration, however, can be affected by factors such as the irrigation protocol, sealer activation, the filling method used, and root canal morphology. EndoSequence BC Sealer showed the highest sealer penetration among the tested sealers. The influence of different fluorochromes on the results of dentinal tubule penetration studies should also be further explored. The in vitro nature of the included studies limits the applicability of the results into the clinical setting. Prospero registration: CRD42022383896