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This study aims to fabricate silk fibroin/calcium sulfate (SF/CS) composites by one-pot synthesis for bone regeneration applications. The SF was harvested from degummed silkworm cocoons, dissolved in a solvent system comprising of calcium chloride:ethanol:water (1:2:8), and then mixed with a stoichiometric amount of sodium sulfate to prepare various SF/CS composites. The crystal pattern, glass transition temperature, and chemical composition of SF/CS samples were analyzed by XRD, DSC, and FTIR, respectively. These characterizations revealed the successful synthesis of pure calcium sulfate dihydrate (CSD) and calcium sulfate hemihydrate (CSH) when it was combined with SF. The thermal analysis through DSC indicated molecular-level interaction between the SF and CS. The FTIR deconvolution spectra demonstrated an increment in the β-sheet content by increasing CS content in the composites. The investigation into the morphology of the composites using SEM revealed the formation of plate-like dihydrate in the pure CS sample, while rod-like structures of α-CSH surrounded by SF in the composites were observed. The compressive strength of the hydrated 10 and 20% SF-incorporated CSH composites portrayed more than a twofold enhancement (statistically significant) in comparison to that of the pure CS samples. Reduced compressive strength was observed upon further increasing the SF content, possibly due to SF agglomeration that restricted its uniform distribution. Therefore, the one-pot synthesized SF/CS composites demonstrated suitable chemical, thermal, and morphological properties. However, additional biological analysis of its potential use as bone substitutes is required. [ABSTRACT FROM AUTHOR]