Abstract A simple procedure for the separation of nonionic detergents and protein is described. Mixtures of the detergent and protein are chromatographed on Sephadex LH-20. Aqueous ethylene glycol (or glycerol) is used as the eluting solvent. In this solvent system, the protein emerges with the void volume of macromolecules of greater than 15,000 molecular weight. The detergent is retained on the column in various monomeric and polymeric forms. In addition, much of the detergent is retained as a tightly bound form that adheres to the nonpolar matrix of the modified Sephadex LH-20. This procedure is both simple and applicable to a number of separations that may be particularly difficult. However, the usefulness of the procedure for a given separation must be determined experimentally; some general guidelines for the application of the technique are given. The investigation of the separation of Lubrol from protein was facilitated by the preparation of labeled Lubrol; preparation of labeled or colored derivatives of the detergents affords a simple determination of detergent concentrations.