The aim of this work was to conduct the hydrothermal synthesis of hydroxyapatite from cuttlebone with the presence of strontium ions (Sr2+) and the subsequent characterization of the samples using different instrumental methods (XRD, FTIR and TGA) to assess the substitution of ions in the structure of hydroxyapatite. Ionic substitutions affect different parameters like crystal cell, crystal size and morphology which change the properties of HAp and subsequently affect the applicability in bone tissue engineering. SEM analysis confirmed that characteristic highly porous interconnected structure of cuttlebone was preserved after hydrothermal synthesis. Qualitative XRD analysis showed that the structure of the synthesized samples consists of HAp with trace amounts of aragonite phase (1-2 %) in samples with 5 and 10 mol.% strontium added. Further processing of the data using the Rietveld refinement method showed that the volume of the crystal lattice of HAp increases with increasing strontium content, which is consistent with theoretical and literature data. The results obtained by FTIR analysis indicate a decrease in the intensity of phosphate, carbonate and hydroxyl groups with the increase of added strontium. With the addition of strontium, the bands of individual groups do not broad, but retain their initial shape. Using TGA analysis, it was found that at lower strontium content, thermal stability improves, but at higher strontium content, the weight loss increases compared to pure HAp.