It is known that enzymes can be activated through lyophilization in the presence of non-buffer salts for use in nonaqueous media. This study focuses on the effect lyophilization has on the secondary structure of an enzyme and how that effect translates to a change in activity. FTIR spectra were taken at various ratios of salt/enzyme formulations and values of α-helix and β-sheet content were obtained as a function of the mass fraction of salt in the final lyophilized preparation. As the salt ratio of the lyophilized preparation increased, more native-like secondary structure was obtained. The more native-like ratio of α-helix to β-sheet was highly correlative to the catalytic efficiency of subtilisin Carlsberg for the transesterification of N-Ac-L-Phe-OEt with 1-PrOH (as reflected in the value of Vmax/Km), with activation over the salt-free lyophilized enzyme preparation of ca. 3,700-fold for the 98% (w/w) salt preparation. The correlation between native-like secondary structure and enzyme activity was extremely high (r = 0.988). To distinguish salt activation from a simple lyoprotection mechanism, identical experiments were performed with trehalose as the excipient. FTIR showed that secondary structure of subtilisin was maintained with high trehalose concentrations, but enzyme activity remained relatively low. The observation that secondary structures of various enzymes used in this study are not substantially different leads to the conclusion that retention of native-like structure for enzymes in salt matrices is a necessary requirement for high enzyme activity, but is not sufficient for activation.