The mechanical properties of human joints (i.e., impedance) are constantly modulated to precisely govern human interaction with the environment. The estimation of these properties requires the displacement of the joint from its intended motion and a subsequent analysis to determine the relationship between the imposed perturbation and the resultant joint torque. There has been much investigation into the estimation of upper-extremity joint impedance during dynamic activities, yet the estimation of ankle impedance during walking has remained a challenge. This estimation is important for understanding how the mechanical properties of the human ankle are modulated during locomotion, and how those properties can be replicated in artificial prostheses designed to restore natural movement control. Here, we introduce a mechatronic platform designed to address the challenge of estimating the stiffness component of ankle impedance during walking, where stiffness denotes the static component of impedance. The system consists of a single degree of freedom mechatronic platform that is capable of perturbing the ankle during the stance phase of walking and measuring the response torque. Additionally, we estimate the platform's intrinsic inertial impedance using parallel linear filters and present a set of methods for estimating the impedance of the ankle from walking data. The methods were validated by comparing the experimentally determined estimates for the stiffness of a prosthetic foot to those measured from an independent testing machine. The parallel filters accurately estimated the mechatronic platform's inertial impedance, accounting for 96% of the variance, when averaged across channels and trials. Furthermore, our measurement system was found to yield reliable estimates of stiffness, which had an average error of only 5.4% (standard deviation: 0.7%) when measured at three time points within the stance phase of locomotion, and compared to the independently determined stiffness values of the prosthetic foot. The mechatronic system and methods proposed in this study are capable of accurately estimating ankle stiffness during the foot-flat region of stance phase. Future work will focus on the implementation of this validated system in estimating human ankle impedance during the stance phase of walking.