Injectable bone substitute (IBS) based on hydroxyapatite, chitosan and streptomycin has been developed successfully. The IBS was made by mixturing 20% w/v hydroxyapatite and varying the chitosan ratio of 60:40, 65:35, 70:30, 75:25 and adding streptomycin as antibiotic substance. The mixture was added with hydroxyl propyl methylcellulose. The synthesis process was steady and no chemical reaction occurred as proven by Fourier Transform Infrared Spectroscopy (FTIR). The in vitro characterization were acidity (pH) and cytotoxicity test (MTT assay), while the physical characterization performed included injectability test, setting time, and morphology. The acidity test showed that the pH samples reached the human normal pH (6.8-7.4) in seven days. The cytotoxicity test proved that the samples were non-toxic. The repasta test showed that the acidity reached the human pH and could release the IBS pasta around 111-150 seconds. The injectability test indicated that IBS had ability to be injected for 95-96%. The setting time in all samples needed 72-166 minutes when it was injected into human bone scaffold model that was able to coat the pore of its scaffold model which proven by Scanning Electron Microscope (SEM) imaging. The pore size of human bone scaffold model was decreased from ±800 μm into ±120 μm. So, IBS pasta based on hydroxyapatite-chitosan-streptomycin in physicochemical and cytotoxicity behaviour is preferable to be applied for spinal tuberculosis cases.