Use of an 125I radioimmunoassay involving antiserum coupled to magnetizable cellulose, after prednisolone-21-phosphate interference is removed by dichloromethane extraction at pH 7.4 and endogenous glucocorticoid interference is removed by selective chemical derivatization with Girard's Reagent T [(carboxymethyl)trimethylammonium chloride hydrazide], allows determination of prednisolone in 10 microL of serum. Results correlate well with those of an established liquid-chromatographic method for separating prednisolone from its metabolites.