Abstract. A wash cycle using an alkaline solution with a dissolved chemical detergent is a standard clean-in-place (CIP) process for cleaning milking systems. However, long-term chemical use may corrode equipment and create difficulties in wastewater treatment. This study investigated the potential for using alkaline electrolyzed oxidizing (EO) water as an alternative to alkaline chemical detergent for removal of microorganisms and adenosine triphosphate (ATP) on milking system materials. Laboratory trials were performed based on a Box-Behnken response surface design to assess the cleaning effect of alkaline EO water on three materials typically used in milking systems: stainless steel, rubber gasket, and polyvinyl chloride (PVC) hose. Results showed that alkaline EO water treatment was generally enhanced with increased treatment time, temperature, and pH, and their interaction effects were also observed in ATP removal. However, treatment time did not have a dominant role in cleaning PVC hose. Response surface models were developed to reliably predict detected microorganisms and relative light units (RLU) on the three materials after alkaline EO water treatment. Based on the response surface models, the three parameters for alkaline EO water cleaning were optimized as treatment time of 10.0 min, temperature of 61.8°C, and pH of 12, after which microorganisms and RLU were nearly undetectable. Alkaline EO water treatment with the optimized parameters had an equivalent or better cleaning ability compared to the commercial detergent, suggesting its potential as a cleaning and bacteria removal agent for milking systems. Keywords: Alkaline electrolyzed oxidizing water, Cleanliness, Milking system, Response surface model.