Introduction: Prostate volume estimation is of great importance for patient evaluation in a urologist's clinical practice. The accuracy and superiority of the techniques used in volume calculation have always been the subject of debate. Therefore, we conducted a comparative analysis between the volumes derived from transrectal ultrasonography (TRUS), multiparametric prostate magnetic resonance imaging (MpMRI), and three-dimensional (3D)-constructed MpMRI images of patients, who underwent retropubic radical prostatectomy at our institution. Methods: The data of patients with preoperative TRUS and MpMRI who underwent radical prostatectomy (Rp) in our clinic between August 2021 and February 2023 were retrospectively reviewed. The prostatectomy specimens were taken to the pathology department without exposure to any fixative and measured with the water displacement method. All axial T2-weighted sequences were segmented by a single surgeon using 3D Slicer (v. 5.6.2) software, and all measurements were compared with the specimen volume measured at the pathology laboratory. Results: A total of 150 patients were included in this study. The median prostate volumes estimated by TRUS-ellipsoid, TRUS-bullet, MpMRI, and 3D segmentation were 43.45 cc (min.-max.: 15.1-122.6), 54.32 cc (min.-max.: 18.9-153.3), 44.05 cc (min.-max.: 15.4-128.9), and 43.11 cc (min.-max.: 14.3-110.6), respectively. The median Rp specimen volume measurement in the pathology department was 42 cc (min.-max.: 12-114). When the measurement techniques were compared between each other, it has been shown that the statistically significant difference was caused by TRUS-bullet measurement. No statistically significant difference between the other three measurement techniques as well as between them and the specimen volume measurements were detected. Conclusion: Consistent with the findings of previous studies, MpMRI has provided estimations closer to pathology measurements and 3D segmentation allows even more precise measurements. However, considering accessibility, reproducibility, time efficiency, and cost, TRUS-based measurements can be safely used in clinical practice, especially using the ellipsoid formula.