Use of dialysate as supplement fluid in hemodiafiltration requires controlling contamination by endotoxin of the dialysate. We thus aimed at developing an endotoxin monitoring system with complete exclusion of endotoxin contamination for simple, easy, and accurate measurement of endotoxin concentration in dialysate. In the present study, we used a 6 valve injector along with a high performance liquid chromatogram system. This new system showed a sensitivity of approximately 1 endotoxin units (EU)/L in the range of 0 to 30 EU/L endotoxin in dialysate and no trace of endotoxin contamination. In conclusion, the new endotoxin monitoring system showed high sensitivity and reproducibility, with easy operation.