Heparin is a naturally occurring highly charged polyanion and a well-known anticoagulant widely used in surgical practice.[1] More specifically, it is a negatively charged sulfated polysaccharide belonging to the group of glycosaminoglycans. The anticoagulant effect of heparin is based on its ability to activate antithrombin-III, which, subsequently, inactivates vital coagulation cascade substance such as thrombin and factor Xa consequently preventing fibrin and clot formation.[2] To balance and neutralize heparin dosing, a heparin inhibitor is needed, and commonly, arginine-rich protein protamine sulfate (PS) is used for this purpose.[3] However, in recent years there has been an interest to investigate alternatives for protamine due to its adverse effects like hypotension and serious allergic reactions.[4] In this study, a host-guest complex capable of selective heparin binding and sensing is presented.[5] Heparin binding affinity was studied in solution with a variety of polycationic macrocyclic hosts, a pillar[5]arene and multiple resorcin[4]arenes, by dynamic light scattering, dye displacement assay, isothermal titration calorimetry, and anti-Xa assay. The measurements reveal the significant importance of multivalency in the electrostatic host-heparin binding in competitive, application-relevant media. Additionally, to monitor heparin concentration, a host-guest indicator displacement assay was performed by following the free and bound state of methyl orange dye in UV-Vis spectroscopic experiments. Furthermore, this colorimetric sensing based on the tertiary host-guest-heparin supramolecular assembly was utilized in the construction of a calibration curve in a range of blood plasma concentrations. Figure 1