Background and objective: During the last 200 years, there have been many changes in the way of performing endodontic treatment. The increased demand from patients for saving their teeth has led to the development of various innovative equipment and advances in the field of material sciences. Thus, the standard protocol of endodontic treatment has undergone several modifications. Bioceramic materials in endodontics can be considered a magnanimous entity with the ability to change the prognosis of many cases, especially those with poor prognosis. Aim: The aim of this in vitro study was to compare and evaluate pH, calcium ion release, and setting time of premixed and freshly mixed tricalcium silicate-based endodontic materials. Materials and methods: Polyethylene tubes measuring 10 mm in length × 1.0 mm in diameter were used. Group I (n = 15)—Polyethylene tubes filled with ProRoot MTA; group II (n = 15)—Polyethylene tubes filled with Biodentine; group III (n = 15)—Polyethylene tubes filled with NeoPutty MTA. Samples were placed in polypropylene flasks containing 10 mL of deionized water. At 6 hours, 12 hours, 24 hours, 7 days, and 1 month, the deionized water was measured for pH using a digital pH meter, and released calcium ions were measured by atomic absorption spectrophotometry. Ten specimens, each with an internal diameter of 10 mm and a thickness of 2 mm, were prepared for each material. A Vicat-type needle with a weight of 100 ± 0.5 gm and a flat end of 2.0 ± 0.1 mm in diameter was used to determine the initial setting time, and another Vicat-type needle with a weight of 456 ± 0.5 gm and a flat end of 1 ± 0.1 mm in diameter was used to determine the final setting time. Results: The results of mean pH values of all three study groups showed that the overall mean pH value of ProRoot MTA (group I) was 10.99 ± 0.782, followed by group II, that is, Biodentine with 10.52 ± 1.108, and the least values were obtained from NeoPutty MTA (group III) with 9.77 ± 1.103, respectively (p-value > 0.001), which shows no statistically significant difference among the three groups. The mean values of calcium release observed in the study groups showed that the highest calcium ion release was shown by ProRoot MTA (group I) with 265.9 ± 136.7, followed by group II, that is, Biodentine with 108.8 ± 49.70, and the least values were obtained from NeoPutty MTA (group III) with 47.84 ± 24.05. All the experimental materials showed Ca²⁺ release at all time periods, with significantly higher calcium ion release observed in group I (ProRoot MTA). However, NeoPutty MTA showed the significantly lowest calcium ion release compared to the other two groups. This difference in the values of calcium ion release across all three groups was found to be statistically significant with a p-value = 0.005. Moreover, on evaluation of setting time, Biodentine showed faster initial and final setting times compared to the other groups. Conclusion: The innovative endodontic cement, ProRoot MTA, exhibited an enhanced alkaline pH value and calcium ion release compared to Biodentine and NeoPutty MTA as bioceramic tricalcium silicate-based endodontic materials.