The cleaning validation is necessary to establish the quality and safety of pharmaceutical drug products. In the cleaning validation protocols, the direct sampling is performed with swabs, which are sticks with textiles at one side. The sample on the swab after the swabbing on the surface of equipment is analyzed with TOC analyzer and HPLC. Recently, HPLC has been more preferable because of the growing need for the individual analysis of products. Before the HPLC analysis, manual processes such as a sample extraction and a sample condensation are required. Such manual processes may affect to the quality of results. Thus, we evaluated the application of a novel on-line supercritical fluid extraction/chromatography system for the cleaning validation.