Reptiles can suffer from infectious and noninfectious cardiac pathologies, requiring the need for standardized diagnostic approaches and reference intervals. Despite the popularity of ball pythons (Python regius) as pets, echocardiographic measurements are unknown in this species. Twenty healthy adult ball pythons were evaluated to identify imaging planes, establish reference intervals for cardiac assessment by two-dimensional echocardiography, and study the effects of sex, body length, and body mass on heart rate, fractional shortening, and vascular, atrial, and ventricular dimensions. Echocardiography was performed under manual restraint. Most cardiac measurements were positively correlated with body length and mass, with the strongest correlation between ventricular end-systolic measurements and body length. The only significant difference found between sexes was for right and left atrial lengths. This study provides guidelines and reference intervals for two-dimensional echocardiographic measurements in adult healthy ball pythons.