Trypsin is a long-known serine protease widely used in biochemical, analytical, biotechnological, or biocatalytic applications. The high biotechnological potential is based on its high catalytic activity, substrate specificity, and catalytic robustness in non-physiological reaction conditions. The latter is mainly due to its stable protein fold, to which six intramolecular disulfide bridges make a significant contribution. Although trypsin does not depend on cofactors, it essentially requires the binding of calcium ions to its calcium-binding site to obtain complete enzymatic activity and stability. This behavior is inevitably associated with a limitation of the enzyme’s applicability. To make trypsin intrinsically calcium-independent, we removed the native calcium-binding site and replaced it with another disulfide bridge. The resulting stabilized apo-trypsin (aTn) retains full catalytic activity as proven by enzyme kinetics. Studies using Ellmann’s reagent further prove that the two inserted cysteines at positions Glu70 and Glu80 are in their oxidized state, creating the desired functional disulfide bond. Furthermore, aTn is independent of calcium ions, possesses increased thermal and functional stability, and significantly reduced autolysis compared to wildtype trypsin. Finally, we confirmed our experimental data by solving the X-ray crystal structure of aTn.