PURPOSE: To obtain an S-substituted alkylated reduced lysozyme useful for the determination of the specificity of protease, by reacting reduced lysozyme with an S-substituted alkylation agent, thereby carrying out the S-substituted alkylation of at least a part of the SH group of the reduced lysozyme.

CONSTITUTION: A reduced lysozyme is produced by cleaving lysozyme with a reducing agent (e.g. mercaptoethanol) and reducing at least a part of the S-S bond to SH group. The reduced lysozyme is made to react with an S- substituted alkylation agent to effect the substituted alkylation of at least a part of the SH group to obtain the objective S-substituted alkylated reduced lysozyme. Concrete examples of the S-substituted alkylation process are carboxymethylation with iodoacetic acid, trimethylaminopropylation with 3- bromopropyltrimethylammonium bromide, etc.