A method for the determination of the effectiveness of gelatin hardeners was proposed. Formaldehyde(FA) and 1,3-bis (vinylsulfonyl)-2-propanol (BVSP) were used as cross-linking agents for the hardening of gelatin. The cross-linked gelatin showed a typical rubber elasticity in a swollen state with a mixed solution composed of concentrated aqueous LiBr and diethylene glycol monoalkyl ether. The energy components to total retractive forces were below 0.3. The amount of intermolecular crosslinks was estimated by applying an usual rubber elasticity theory for the swollen samples. The number of cross-linking sites was also determined by the amino acid analysis of BVSP cross-linked gelatin. The cross-linking efficiency for the reaction of BVSP occurring in a casting film with less swelling conditions was about four times higher than in a swollen gel. Approximately 92% or more of BVSP used was reacted with the free amino groups of lysine and arginine residues. There was a significant difference between the reactivities of these amino groups. The amino groups of lysine residues were more reactive than those of arginine in a gel state; the reactivities, however, reversed in a thin layer of wet film. The effectiveness of FA was extremely low.