An enzyme immunoassay for the measurement of thymosin beta 4, a chemically characterized thymic polypeptide, is described. A limited amount of reagent antiserum to synthetic thymosin beta 4 is incubated with liquid-phase thymosin beta 4. Unbound antibody is then incubated with solid-phase thymosin beta 4. The method is specific, sensitive, highly reproducible and capable of detecting as little as 2.5 ng/ml of thymosin beta 4.