Due to its pronounced regenerative capacity, the zebrafish heart represents an advantageous model system for exploring the cellular and molecular mechanisms of cardiac regeneration. Upon injury, the epicardium, the outermost mesothelial tissue layer of vertebrate hearts, serves dual purposes in the regenerating heart as both a signaling center and a source for crucial cell types. Traditional in vivo genetic approaches to study heart regeneration can be time consuming and are not applicable to large-scale approaches and live surveillance of cellular behaviors. Here, we demonstrate ex vivo methods to culture, maintain, and study the regenerative responses of epicardial tissue in excised zebrafish hearts. Epicardial cell proliferation and migration are monitored in real time after uninjured or injured hearts are excised, washed, and cultured for up to 30 days. In addition to these techniques, we describe ex vivo genetic ablation of the epicardium, cell proliferation assays, partial ventricular explant culturing, and chemical screening.