The recent global COVID-19 pandemic caused by SARS-CoV-2 lasted for over three years. A key measure in combatting this pandemic involved the measurement of the monoclonal antibody (mAb)-mediated inhibition of binding between the spike receptor-binding domain (RBD) and hACE2 receptor. Potency assessments of therapeutic anti-SARS-CoV-2 mAbs typically include binding or cell-based neutralization assays. We assessed the inhibitory activity of five anti-SARS-CoV-2 mAbs using ELISA, surface plasmon resonance (SPR), and four cell-based neutralization assays using different pseudovirus particles and 293T or A549 cells expressing hACE2 with or without TMPRSS2. We assessed the interchangeability between cell-based and binding assays by applying the Bland–Altman method under certain assumptions. Our data demonstrated that the IC₅₀ [nM] values determined by eight neutralization assays are independent of the cell line, presence of TMPRSS2 enzyme on the cell surface, and pseudovirus backbone used. Moreover, the Bland–Altman analysis showed that the IC₅₀ [nM] and K_D [nM] values determined by neutralization/ELISA or by SPR are equivalent and that the anti-spike mAb activity can be attributed to one variable directly related to its tertiary conformational structure conformation, rate dissociation constant K_off. This parameter is independent from the concentrations of the components of the mAb:RBD:hACE2 complexes and can be used for a comparison between the activities of the different mAbs.