||Various natural polymers are used in biomedical fields due to its biocompatibility and biodegradability. Among them, silk proteins can be designed into various forms including hydrogels, films, porous scaffolds, and fibers. Regenerated silk fibroin (RSF) hydrogels are stabilized due to the thermodynamically stable β-sheet structure which can be induced by temperature, ionic strength and pH of the solution. In this study, we prepared RSF hydrogels via slow acidification by adding glucono-delta-lactone (GDL). As GDL slowly hydrolyzed into gluconic acid and lowered the pH of the RSF solution, the β-sheet transition of RSF proteins occurs and finally RSF hydrogel were formed. In addition, RSF hydrogels by fast acidification were prepared for comparison by adding 1M HCl. For the characterization, FE-SEM, FT-IR, and UTM were performed for both RSF hydrogels. Furthermore, gelation behavior was confirmed by UV-Vis spectrometry and circular dichroism analysis.