Chitosan (CS) has special properties such as biocompatibility, biodegradability, antibacterial, and biological activity which make this material is currently studied in various applications, including tissue engineering. There are different methods to modify the morphology of CS. Most use chemical crosslinking agents, however, those methods have disadvantages such as low polymer degradability and unwanted side effects. The objective of this research was to obtain CS spheres through the physical crosslinking of commercial CS without using crosslinking agents through a simple coacervation method. A central composite experimental design was used to optimize the synthesis of the CS spheres and by the response surface methodology it was possible to obtain CS spheres with the smallest diameter and the most regular morphology. With the optimal formulation (CS solution 1.8% (w/v), acetic acid (AAC) solution 1% (w/v), sodium hydroxide (NaOH) solution 13% (w/v), relative humidity of (10%) and needle diameter of 0.6 mm), a final sphere diameter of 1 mm was obtained. Spheres were characterized by physical, chemical, thermal, and biological properties in simulated body fluid (SBF). The results obtained allowed us to understand the effect of the studied variables on the spheres’ diameter. An optimized condition facilitated the change in the morphology of the CS while maintaining its desirable properties for use in tissue engineering.