Two major modifications of existing methods for the quantitative estimation of serum bile salts by gas–liquid chromatography have been made. A new protein precipitation step using 2,2-dimethoxypropane gives almost complete recovery of added bile salts. A purification step using thin-layer chromatography gives good recovery of mono-, di-, and tri-hydroxy bile acids. Overall recovery of bile salts is greater than 70%. The new procedure reduces the time required for serum bile salt analysis.