Purpose: The purpose of this study was to develop nursing practice guidelines for water treatment system used in hemodialysis and to evaluate the guidelines by applying them in practice. Method: The first draft for the guidelines was developed based on advice and recommendations obtained from procedure review of critical literature. The draft was modified through evaluation by an expert group and pilot application to practice. The final draft was evaluated by the expert group using the AGREE instrument (Appraisal of Guidelines for Research and Evaluation). Results: For the pilot test using the draft guidelines, 144 samples were collected from the water treatment system and hemodialysis machines. Results showed no bacteria. Endotoxin tests and chemical tests passed the criteria. After revision of the draft guidelines and additions to the draft guidelines, the final draft was confirmed. The quality of the final draft was evaluated by 4 experts using the AGREE instrument. The mean standard score was 76.9% for the 19 items. Conclusion: The clinical guidelines developed in this research can be utilized as systematic and scientific guidelines for water treatment systems used in hemodialysis. In addition, the results of the research can contribute to improving care services.