Background: Phosphate is an important factor in explaining the high progress of vascular calcification among dialysis patients. Today, phosphate concentration is measured in plasma on a regular basis. The aim of this study was to find out if it is possible to estimate total removed phosphate (TRp) in spent dialysate utilizing UV absorbance during hemodiafiltration. Methods: Eleven patients were monitored online with UV absorbance at 297 nm, three times during one week each (n = 33). Dialysate samples were taken at different times during treatment and from a collection tank to chemically determine phosphate concentrations. Two mathematical models (UVIND and UVGROUP) were tested to estimate TRp with supervision by UV absorbance and compared with TRp measured in the tank (reference). Results: High correlation between UV absorbance and phosphate concentration for each single patient and lower for the whole group together was found. TRp was (mean ± SD) 30.7 ± 7.3 mmol for the reference and 30.8 ± 8.2 and 29.1 ± 5.2 mmol for UVIND and UVGROUP, respectively (p > 0.05). Conclusion: This study demonstrates a novel possibility to estimate TRp based on linear relationship between online monitoring of UV absorbance and concentration of phosphate in spent dialysate.