Objective  To develop a single high-performance liquid chromatography (HPLC) method for simultaneous determination of acetate and citrate in hemodialysis solution and concentrate, thereby improve detection efficiency, reduce cost, and simplify operation the procedure.  Methods  A C18 column (octadecyl-bonded monodisperse porous silica microspheres) was used with 50 mmol/L phosphate buffer (pH 2.0) as the mobile phase. The flow rate was 1.0 mL/min, the detection wavelength was 210 nm, and the column temperature was 30℃. The contents of acetate and citrate in samples were determined. Results Acetate was linear in the range of 1～20 mmol/L(r=0.999957), with recoveries of 99.07%～103.41% and RSD of 2.37% (n=3). Citrate showed good linearity in the range of 0.1～15 mmol/L(r=0.999966), with recoveries of 101.71%～104.49% and RSD of 1.42%(n=3). Conclusion  This single HPLC method is simple, accurate, and suitable for the simultaneous determination of acetate and citrate in hemodialysis solution and concentrate. It can be applied to quality control during production and clinical use, thereby effectively improving detection efficiency.