Macromolecular association is crucial to many fields in biomedical sciences, including drug development, gene editing, and diagnostics. In particular, protein−protein association and dissociation rate constants are typically determined using surface plasmon resonance systems, which require costly instrumentation and cumbersome procedures (e.g., blocking, washing, and separation). Herein, we demonstrate that protein-binding constants can be readily determined using a real-time biosensing platform facilitated by graphene oxide-modified microwell plates and fluorophore-labeled proteins, where the fluorescent probes remain highly fluorescent during protein association, whereas fluorescent bioprobes that are not associated with their counterparts are quenched by graphene oxide. Binding data of three pairs of proteins were systematically determined employing this single-step platform and compared with those data reported by the suppliers or the literature, suggesting that this approach is comparable and consistent with the existing ones. Such pairs include (i) human immunoglobulin G (H-IgG)−fluorophore-labeled anti-H-IgG, (ii) prostate-specific antigen (PSA)−quantum dot-labeled anti-PSA, and (iii) anti-RBD−fluorophore-labeled SARS-CoV-2 spike receptor-binding domain recombinant protein. We also offer an opensource software that automatically determines the binding kinetics constants of proteins. This Technical Note introduces a simple, yet effective, platform to determine relevant information on protein kinetics, which can be performed using a microwell plate reader and economical materials like graphene oxide. We foresee a new generation of diagnostics based on our affordable protein kinetics analysis.