Walking is the primary mode of human locomotion. Accordingly, people have been interested in studying human gait since at least the fourth century BC. Human gait analysis is now common in many fields of clinical and basic research, but gold standard approaches – e.g., three-dimensional motion capture, instrumented mats or footwear, and wearables – are often expensive, immobile, data-limited, and/or require specialized equipment or expertise for operation. Recent advances in video-based pose estimation have suggested exciting potential for analyzing human gait using only two-dimensional video inputs collected from readily accessible devices (e.g., smartphones, tablets). However, we currently lack: 1) data about the accuracy of video-based pose estimation approaches for human gait analysis relative to gold standard measurement techniques and 2) an available workflow for performing human gait analysis via video-based pose estimation. In this study, we compared a large set of spatiotemporal and sagittal kinematic gait parameters as measured by OpenPose (a freely available algorithm for video-based human pose estimation) and three-dimensional motion capture from trials where healthy adults walked overground. We found that OpenPose performed well in estimating many gait parameters (e.g., step time, step length, sagittal hip and knee angles) while some (e.g., double support time, sagittal ankle angles) were less accurate. We observed that mean values for individual participants – as are often of primary interest in clinical settings – were more accurate than individual step-by-step measurements. We also provide a workflow for users to perform their own gait analyses and offer suggestions and considerations for future approaches.