Abstract—The time‐resolved tryptophyl fluorescence of α‐chymotrypsinogen A and α‐chymotrypsin in the crystalline state and in buffer solution at room temperature was analyzed globally. Tripleexponential decay functions are necessary to adequately describe the tryptophyl fluorescence decay surfaces of the protein powders as a function of hydration and in solution. The fluorescence lifetimes of α‐chymotrypsinogen A (τ1= 0.32 ns. τ2= 1.30 ns. τ3= 3.98 ns) and α‐chymotrypsin (τ1= 0.66 ns. τ2= 2.26 ns. τ3= 5.40 ns) are constant over the entire hydration range. The spectral positions of the decay‐associated spectra of the hydrated powders do not shift as a function of hydration. This indicates that the structures of the zymogen and the active enzyme are unaffected by hydration. The lifetimes of α‐chymotrypsinogen A in phosphate buffer pH 7.4 are τ1= 0.37 ns, τ2= 1.17 ns and τ3= 3.44 ns while the respective values of α‐chymotrypsin are τ1= 0.4