Colloid osmotic pressure (COP) is important in calculating vascular permeability during hemodialysis (HD). However, few reports have directly measured COP in HD patients. Therefore, the currently published formulas may not be clinically optimal for predicting COP for HD patients. Here, the study aims were (i) to directly measure COP in HD patients and compare the measured and predicted COP values using four previously reported formulas, and (ii) to develop a formula for approximating COP using clinical parameters. We obtained 212 measured COP values using an osmometer; the average value was 22.0 ± 0.2 mm Hg. The predicted COP based on the four different formulas was positively correlated with the measured COP (0.87 < r < 0.89), but was significantly overestimated compared to it (P < 0.001). We also performed a stepwise analysis using serum albumin and non-albumin protein concentrations and obtained the following simple formula for COP approximation: COP (mm Hg) = -7.91 + 5.64 × albumin (g/dL) + 3.00 × non-albumin proteins (g/dL). A positive linear correlation was observed between the measured COP and approximated COP using this formula (r = 0.90, P < 0.001). We calculated the mean Kr (plasma-refilling coefficient) as a marker for determining dry weight in HD patients using the measured COP and approximated COP. No differences were observed between the mean Kr derived from the measured and approximated COP. We report here significant differences between measured and predicted COP values, and have devised a simple formula for COP approximation in HD patients.