During natural locomotion, the stiffness of the human knee is modulated continuously and subconsciously according to the demands of activity and terrain. Given modern actuator technology, powered transfemoral prostheses could theoretically provide a similar degree of sophistication and function. However, experimentally quantifying knee stiffness modulation during natural gait is challenging. Alternatively, joint stiffness could be estimated in a less disruptive manner using electromyography (EMG) combined with kinetic and kinematic measurements to estimate muscle force, together with models that relate muscle force to stiffness. Here we present the first step in that process, where we develop such an approach and evaluate it in isometric conditions, where experimental measurements are more feasible. Our EMG-guided modeling approach allows us to consider conditions with antagonistic muscle activation, a phenomenon commonly observed in physiological gait. Our validation shows that model-based estimates of knee joint stiffness coincide well with experimental data obtained using conventional perturbation techniques. We conclude that knee stiffness can be accurately estimated in isometric conditions without applying perturbations, which presents an important step toward our ultimate goal of quantifying knee stiffness during gait.