The ÄKTA pilot 600 chromatography system is designed to facilitate sanitization, with the high standards imposed by regulatory authorities for purity of the clinical products in mind. In this study, the predefined sanitization method developed for ÄKTA pilot 600 was assessed. In a microbial challenge test, parts were exposed to a bacterial strain recommended by the United States Pharmacopoeia (USP 38) and to yeast strains commonly used in production environments. Sanitization of the system was performed by running the predefined method using 1 M sodium hydroxide (NaOH) as sanitization agent. The results show excellent cleaning efficiency, with a log reduction factor of ≥ 6 for colony forming units (CFU) of the test microorganisms. Sanitization of the SNAP-connectors using 1 M NaOH was also successfully conducted.