The investigation started with an experimental heparin binding competition assay (HBCA) exploiting Mallard Blue (MB), a deep-blue colored, positively charged (+5) synthetic dye based on an arginine-functionalized thionine. This compound was specifically developed by our group for ultra-high-affinity sensing of clinically-relevant heparin levels in both physiological solutions and serum [34]. The dye reports on heparin levels by quantitatively binding to the polyanion; this, in turn, results in a significant change in its UV−visible spectroscopic profile. Based on this evidence, we conceived the HBCA as follows: First, two solutions containing heparin and MB are mixed and then the resulting system is gradually titrated with solutions containing protamine and PAMAM dendrimers of varying generations. In the presence of increasing concentration of each possible heparin binder, the dye is gradually displaced from heparin and the absorbance intensity of free MB increases accordingly, as shown in Figure 2a. Curve fitting of the data shown in this figure allowed us to calculate the main binding parameters, i.e., the effective heparin binder concentration require to displace 50% of the MB dye from the polyanion (EC50), and the charge excess, that is the number of binder positive charges per heparin negative charge (CE50) required to attain 50% displacement of the MB dye. These values are listed in Table A1.