Three cases of metabolic bone disease (MBD) were identified in juvenile Humboldt penguins (Spheniscus humboldti) in a zoological collection. Diagnosis, monitoring, and treatment were challenging, in part because radiographs and traditional serum biochemistries did not provide adequate information to guide appropriate clinical management. Normal values for ionized calcium (iCa), 25-hydroxyvitamin D₃ [25-(OH) D₃], and parathyroid hormone (PTH) have not been reported for any species in the order Sphenisciformes. This study aimed to establish reference ranges for these parameters to provide a method for assessing clinical cases of MBD and other disease processes. iCa was measured in 33 healthy adult birds from two zoological collections by using a portable clinical analyzer. iCa also was measured from 14 of these birds at a commercial laboratory. Mean and standard deviation were determined to be 1.21 ± 0.09 and 1.29 ± 0.10 mmol/L, respectively. Limited data exist on iCa in avian species, but these results are consistent with other reports and provide a useful clinical parameter. Analysis of PTH and 25-(OH) D₃ was performed at a commercial laboratory on samples from 14 healthy adult penguins in one collection. Means and standard deviations for PTH and 25-(OH) D₃ were 0.8 ± 0.3 pmol/L and 3.7 ± 2.4 nmol/L, respectively. These results are near the minimal functional detectable limits of the assays; raising uncertainty about the validity and usefulness of currently available PTH and 25-(OH) D₃ tests in this species.