Introduction: The aim of this study was to evaluate the effect of previous root canal treatment on dentinal tubule penetration of calcium silicate–based sealers during endodontic retreatment and to compare 2 different (commonly used and modified) tubule penetration depth measurement techniques. Methods: The crowns of mandibular premolar teeth were removed, and root canals were prepared (ProTaper Universal; Dentsply Sirona, York, PA) and obturated using AH Plus (AH; Dentsply DeTrey, Konstanz, Germany), MTA Fillapex (MTAF; Angelus, Londrina, Brazil), and WellRoot ST (WRST; Vericom, Gyeonggi-do, South Korea); 0.1% red Rhod-2 dye (Chemodex, St Gallen, Switzerland) was added to the sealers. After storage for 3 weeks (37°C, 100% humid), the following sealer combination subgroups were created (the sealer used during the primary root canal treatment/the sealer used during the retreatment, n = 8): AH/AH, AH/MTAF, and AH/WRST; MTAF/AH, MTAF/MTAF, and MTAF/WRST; and WRST/AH, WRST/MTAF, and WRST/WRST. Green Fluo-3 dye (0.1%) was added to the sealers during retreatment. Sealer penetration depths were measured at 8 points and averaged, and the ratio of the retreatment sealer to the initial sealer was recorded as the mean tubule penetration depth ratio in the first technique, whereas the percentage of the circumferential penetration area ratios of the retreatment and initial sealers was calculated in the second technique. Statistical analyses were performed using Kolmogorov-Smirnov, Kruskal-Wallis, and Mann-Whitney U tests. Results: The mean penetration depth of AH and MTFA was found to be higher at the coronal third region in the control groups (P < .05). Both methods showed a significant difference among the groups at the coronal and median regions (P < .05). The AH/WRST, MTAH/AH, MTAF/MTAF, MTAF/WRSTS, and WRST/WRST groups showed deeper tubule penetration (first technique, P = .01). Both techniques showed that the penetration depth of the sealers was the same at the apical region (P > .05), whereas a difference was found among the techniques at the coronal and median regions. The sealers used in the previous root canal treatment limited the penetration depth of the sealers used during the retreatment process (P < .05). When MTAF was used as the initial sealer, the penetration depth of the second sealer did not change, whereas if WRST was the initial sealer, the penetration depth of the sealers at the median and apical regions is negatively affected during the retreatment process (P < .05). Conclusions: Previous root canal treatment affects the penetration depth of calcium silicate–based sealers. The use of AH Plus and MTA Fillapex during the retreatment process showed an advantage. Both measurement techniques showed similar performances at the apical region, whereas the results varied at the coronal and midcoronal regions; therefore, a more stable sealer penetration measurement technique is still necessary.