There are challenges associated with design and development of affinity biosensors due to the complicated multiphysics nature of the system. Understanding the binding interaction between target molecules and immobilized receptors and its kinetics is a crucial step to develop robust and reliable biosensor technologies. Evaluation of binding kinetics in biosensors becomes more important and challenging for clinical samples with a complex matrix. Despite drastic advancements in biosensor technologies, having a practical perception of the binding kinetics has remained a critical bottleneck due to limited fundamental understanding. This Review aims to provide a comprehensive discussion on concepts and advances developed so far for the perception of binding kinetics in affinity biosensors. Here, modeling approaches and measurement techniques are presented to characterize the binding interactions in biosensor technologies, while the effect of fouling and secondary factors in the binding interactions will be discussed in the concept of kinetics. This Review will investigate the existing research gaps and potential opportunities in the perception of binding kinetics and challenges to develop robust and reliable biosensors.