Background: 223Ra imaging is crucial to evaluate the successfulness of the therapy of bone metastasis of castration-resistant prostate cancer (CRPC). The goals of this study were to establish a quantitative tomographic 223Ra imaging protocol with clinically achievable conditions, as well as to investigate its usefulness and limitations. We performed several experiments using the Infinia Hawkeye 4 gamma camera (GE) and physical phantoms in order to assess the optimal image acquisition and reconstruction parameters, such as the windows setting, as well as the iteration number and filter of the reconstruction algorithm. Then, based on the MIRD pamphlet 23, we used a NEMA phantom and an anthropomorphic TORSO® phantom to calibrate the gamma camera and investigate the accuracy of quantification. Results: Experiences showed that the 85 keV ± 20%, 154 keV ± 10%, and 270 keV ± 10% energy windows are the most suitable for 223Ra imaging. The study with the NEMA phantom showed that the OSEM algorithm with 2 iterations, 10 subsets, and the Butterworth filter offered the best compromise between contrast and noise. Moreover, the calibration factors for different sphere sizes (26.5 ml, 11.5 ml, and 5.6 ml) were constant for 223Ra concentrations ranging between 6.5 and 22.8 kBq/ml. The values found are 73.7 cts/s/MBq, 43.8 cts/s/MBq, and 43.4 cts/s/MBq for 26.5 ml, 11.5 ml, and 5.6 ml sphere, respectively. For concentration lower than 6.5 kBq/ml, the calibration factors exhibited greater variability pointing out the limitations of SPECT/CT imaging for quantification. By the use of a TORSO® phantom, we simulated several tumors to normal tissue ratios as close as possible to clinical conditions. Using the calibration factors obtained with the NEMA phantom, for 223Ra concentrations higher than 8 kBq/ml, we were able to quantify the activity with an error inferior to 18.8% in a 5.6 ml lesion. Conclusions: Absolute quantitative 223Ra SPECT imaging appears feasible once the dimension of the target is determined. Further evaluation should be needed to apply the calibration factor-based quantitation to clinical 223Ra SPECT/CT imaging. This will open the possibility for patient-specific 223Ra treatment planning and therapeutic outcome prediction in patients.