Wild populations of Humboldt penguins (Spheniscus humboldti) on the coasts of Chile and Peru have been declining because of food scarcity caused by the El Niño Southern Oscillation and human interference. Part of conserving this vulnerable and threatened species is maintaining the health of penguins within zoo collections. A variety of cardiovascular diseases has been reported in individuals from the Spheniscidae family including ventricular septal defects, Dirofilaria immitis infection, pulmonary hypertension, and valvular dysplasia, ultimately resulting in congestive heart failure. An accurate clinical picture of cardiovascular disease in Humboldt penguins requires diagnostics tailored to this specific species. The aim of this study was to establish a routine methodology for evaluating the cardiac silhouette of clinically healthy Humboldt penguins using vertebral heart scale (VHS), cardiocoelomic width ratio (CCWR), and a novel cardiac silhouette-to-keel ratio (CKR). Ventrodorsal and right lateral radiographs were taken of 10 mature Humboldt penguins during routine health evaluations. An echocardiographic exam of each penguin was performed to confirm that there was no evidence of cardiac structural remodeling from disease. Two penguins were excluded on the basis of echocardiographic findings; therefore, data from eight penguins were used to calculate objective cardiac measurements for the clinically healthy population. Right lateral radiographs were used to determine VHS (7.4–10.4) and CKR (3.4–4.4). Ventrodorsal radiographs were used for calculating CCWR (0.45–0.59). The excluded penguins had CCWRs that were outside the 95% confidence interval for the range generated by this study. This initial work supports that standardizing radiographic views provides objective measures for cardiac silhouette evaluation in this species. Further research in larger populations and comparison with birds having confirmed cardiac disease is needed to determine the value of these three measurement techniques in Humboldt penguins.