Calcium-dependent cardiac muscle contraction is regulated by the protein complex troponin (cTn) and specifically by the regulatory N-terminal domain (N-cTnC) which contains one active Ca2+ binding site (site II). It has been previously shown that cardiac muscle contractility and functionality is affected by mutations in N-cTnC which alter calcium binding affinity. Here, we describe the application of adaptive steered molecular dynamics to characterize the influence of N-cTnC mutations on site II calcium binding affinity. We observed the correct trends for all of the studied calcium sensitizing and desensitizing mutants, in conjunction with loop II perturbations. Additionally, the potential of mean force accuracy was shown to increase substantially with increasingly slower speeds and using fewer trajectories. This study presents a novel approach to computationally estimate the Ca2+ binding affinity of N-cTnC structures and is a valuable potential tool to support the design and characterization of novel mutations with potential therapeutic benefits.