ABSTRACT Background Kt/Vurea is the most used marker to estimate dialysis adequacy; however, it does not reflect the removal of many other uraemic toxins, and a new approach is needed. We have assessed the feasibility of estimating intradialytic serum time-averaged concentration (TAC) of various uraemic toxins from their spent dialysate concentrations that can be estimated non-invasively online with optical methods. Methods Serum and spent dialysate levels and total removed solute (TRS) of urea, uric acid (UA), indoxyl sulphate (IS) and β2-microglobulin (β2M) were evaluated with laboratory methods during 312 haemodialysis sessions in 78 patients with four different dialysis treatment settings. TAC was calculated from serum concentrations and evaluated from TRS and logarithmic mean concentrations of spent dialysate (MlnD). Results Mean (± standard deviation) intradialytic serum TAC values of urea, UA, β2M and IS were 10.4 ± 3.8 mmol/L, 191.6 ± 48.1 µmol/L, 13.3 ± 4.3 mg/L and 82.9 ± 43.3 µmol/L, respectively. These serum TAC values were similar and highly correlated with those estimated from TRS [10.5 ± 3.6 mmol/L (R2 = 0.92), 191.5 ± 42.8 µmol/L (R2 = 0.79), 13.0 ± 3.2 mg/L (R2 = 0.59) and 82.7 ± 40.0 µmol/L (R2 = 0.85)] and from MlnD [10.7 ± 3.7 mmol/L (R2 = 0.92), 191.6 ± 43.8 µmol/L (R2 = 0.80), 12.9 ± 3.2 mg/L (R2 = 0.63) and 82.2 ± 38.6 µmol/L (R2 = 0.84)], respectively. Conclusions Intradialytic serum TAC of different uraemic toxins can be estimated non-invasively from their concentration in spent dialysate. This sets the stage for TAC estimation from online optical monitoring of spent dialysate concentrations of diverse solutes and for further optimization of estimation models for each uraemic toxin.