A spectrophotometric method for the determination of cystine with o-phthalaldehyde (OPA) in the absence of thiol is described. When cystine is heated at 60[ddot]C for 30 min in a low excess of OPA (pH 9.5), a very stable derivative with 1:2 stoichiometry (cystine:OPA) and an absorption maximum at 335 nm (E = 4600) is formed. At pH < 1 the derivative is protonated (protonation constants: log K1= 5.88 and log K2= 3.70 at I = 0.1 and 20[ddot]C) and another absorption band at 440 nm (E = 3800) appears, which allows the determination of cystine in the presence of other amino acids.