For most therapeutic antibodies, the interstitium is the target space. Although experimental methods for measuring antibody pharmacokinetics (PK) in this space are not well established, making quantitative assessment difficult, the interstitial antibody concentration is assumed to be low. Here, we combined direct quantification of antibodies in the interstitial fluid with a physiologically‐based PK (PBPK) modelling approach with the goal of better describing the PK of monoclonal antibodies in the interstitial space of different tissues. We isolated interstitial fluid by tissue centrifugation, and conducted an antibody biodistribution study in mice, measuring total tissue‐ and interstitial concentrations in selected tissues. Residual plasma, interstitial volumes and lymph flows, which are important PBPK model parameters, were assessed in vivo. We could thereby refine PBPK modelling of monoclonal antibodies, better interpret antibody biodistribution data and more accurately predict their PK in the different tissue spaces. Our results indicate that in tissues with discontinuous capillaries (liver and spleen), interstitial concentrations are reflected by plasma concentration. In tissues with continuous capillaries (e.g. skin and muscle), ∼50‐60% of plasma concentration is found in the interstitial space. In brain and kidney, on the other hand, antibodies are restricted to the vascular space. Our data may significantly impact the interpretation of biodistribution data of monoclonal antibodies and might be important when relating measured concentrations to a therapeutic effect. Opposing the view that antibodies distribution to the interstitial space is limited, we show by direct measurements and model‐based data interpretation that high antibody interstitial concentrations are reached in most tissues. This article is protected by copyright. All rights reserved