Heparin is a sulfated glycosaminoglycan that is routinely used as an anticoagulant. It is typically purified from bovine or porcine sources, leading to heterogeneity that poses several challenges when used clinically. We have found that the bacteriophage Qβ can be selectively sulfated to yield virus-like nanoparticles (sulf-VLP) that elicit anticoagulant activity similar to heparin. In an effort to explore the binding interactions that heparin-like VLPs make with cationic targets, described herein are bio-layer interferometry studies utilizing the BLItz platform that evaluate the interaction of sulf-VLP with the cationic peptide CDK5 (50 % Lys). Streptavidin biosensors modified with biotin-CDK5 were found to bind strongly to sulf-VLP and not to the underivatized nanoparticle. Titration of sulf-VLP yielded concentration-dependent sensorgrams, permitting calculation of rate and equilibrium constants: k on = (8 ± 3) × 106 s−1 for the association phase, k off = (5 ± 2) × 10−3 M s−1 for the dissociation phase, yielding an overall dissociation constant K D ~ 1 nM. Fitting was best achieved using an equation possessing both exponential and linear terms, suggesting a mechanism more complex than 1:1 binding. To mitigate multivalency and rebinding effects, experiments were conducted with protamine (~70 % Arg) added during the dissociation phase, leading to more pronounced dissociation curves and k off values that yielded a near-linear relationship with protamine concentration.