Background: Sealer penetration increases the contact area between the dentinal walls and the sealer, thus improving the sealing and antimicrobial efficacy of the sealers. Objectives: Evaluation the maximum depth and percentage of AH Plus, Endoseal MTA and Bio-C sealer penetration into the dentinal tubules using single cone and warm vertical compaction (WVC) obturation techniques. Materials and methods: Ninety single rooted mandibular premolars decoronated at 13mm from apex. After instrumentation, samples were randomly divided into 6 groups (n=15) according to the sealers and obturation techniques used. The obturated samples were then kept in 100% humidity at 37C for 7 days and sectioned at 3mm, 5mm and 7mm from the terminus of filling. The sectioned samples were observed under confocal laser scanning microscope to determine maximum penetration depth and percentage of sealer penetration. Statistical analyses was performed using one way ANOVA with Tukey Post-HOC test for multiple comparison and independent t-test. Results: At all the levels, the maximum depth and percentage of penetration of Bio-C and Endoseal MTA were significantly better (p0.05) as compared to AH Plus when the samples were obturated with single cone and warm vertical compaction obturation techniques. However, Endoseal MTA and Bio-C sealers showed no statistically significant difference (p>0.05). Irrespective of the sealer used, warm vertical compaction obturation technique showed a statistically significant (p0.05) difference in depth and percentage of sealer penetration at all the levels. Conclusion: Bio-ceramic sealer (Endoseal MTA and Bio-C) had greater depth and percentage of sealer penetration as compared to AH Plus with both obturation techniques at all the evaluated levels. Furthermore, warm vertical compaction obturation technique achieved greater depth and percentage of sealer penetration as compared to single cone technique.