PurposePositron emission tomography imaging of zirconium-89-labelled monoclonal antibodies (89Zr-Immuno-PET) allows for visualisation and quantification of antibody uptake in tumours in vivo. Patlak linearization provides distribution volume (VT) and nett influx rate (Ki) values, representing reversible and irreversible uptake, respectively. Standardised uptake value (SUV) and tumour-to-plasma/tumour-to-blood ratio (TPR/TBR) are often used, but their validity depends on the comparability of plasma kinetics and clearances. This study assesses the validity of SUV, TPR and TBR against Patlak Kifor quantifying irreversible89Zr-Immuno-PET uptake in tumours.MethodsTen patients received 37 MBq 10 mg89Zr-anti-EGFR with 500 mg/m2unlabelled mAbs. Five patients received two doses of 37 MBq89Zr-anti-HER3: 8–24 mg for the first administration and 24 mg–30 mg/kg for the second. Seven tumours from four patients showed89Zr-anti-EGFR uptake, and 18 tumours from five patients showed89Zr-anti-HER3 uptake. SUVpeak,TPRpeakand TBRpeakvalues were obtained from one to six days p.i. Patlak linearization was applied to tumour time activity curves and plasma samples to obtain Ki.ResultsFor89Zr-anti-EGFR, there was a small variability along the linear regression line between SUV (− 0.51–0.57), TPR (− 0.06‒0.11) and TBR (− 0.13‒0.16) on day 6 versus Ki. Similar doses of89Zr-anti-HER3 showed similar variability for SUV (− 1.3‒1.0), TPR (− 1.1‒0.53) and TBR (− 1.5‒0.72) on day 5 versus Ki. However, for the second administration of89Zr-anti-HER3 with a large variability in administered mass doses, SUV showed a larger variability (− 1.4‒2.3) along the regression line with Ki, which improved when using TPR (− 0.38–0.32) or TBR (− 0.56‒0.46).ConclusionSUV, TPR and TBR at late time points were valid for quantifying irreversible lesional89Zr-Immuno-PET uptake when constant mass doses were administered. However, for variable mass doses, only TPR and TBR provided reliable values for irreversible uptake, but not SUV, because SUV does not take patient and mass dose-specific plasma clearance into account.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00259-023-06151-1.