A direct injection high-performance liquid chromatographic method is developed for the determination of cholesterol in egg-yolk. The described method involved an on-line sample clean-up procedure using column-switching technique and protein-coated RP-18 precolumn. The egg-yolk was diluted with tris buffer, pH 8.5 to produce homogenous sample and render it suitable for injection. On the precolumn, cholesterol was preseparated from endogenous components of the egg-yolk with tris buffer, pH 8.5 for 4 minutes. To complete the precolumn washing cycle, the solvent selector is automatically changed from tris buffer to 0.05 M phosphate buffer, pH 4.8 for 2 min, followed by 15% methanol in 0.05 M phosphate buffer, pH 4.8 for 1 min further.