This study aimed to compare the venous acid-base status of healthy awake versus anesthetized Magellanic penguins (Spheniscus magellanicus). Ten nonanesthetized penguins were manually restrained, and a venous blood sample was collected. Six of these penguins were anesthetized by 2% isoflurane and, after an anesthetic stabilization period, both venous and arterial blood samples were simultaneously withdrawn. Using an i-STAT analyzer, partial pressure of carbon dioxide (PCO₂), partial pressure of oxygen (PO₂), pH, standard bicarbonate concentration (HCO₃^–), total carbon dioxide (ctCO₂), oxygen saturation (SO₂), base excess (BE), Na⁺, and K⁺ levels were measured in venous blood samples of awake (G_awake) penguins and in venous (G_ven) and arterial blood (G_art) samples of anesthetized penguins. There were no significant differences between groups in pH, BE, or Na⁺. Venous carbon dioxide pressure, HCO₃^–, and venous ctCO₂ were higher in G_ven than G_awake penguins, whereas PCO₂ was higher in G_ven than G_art penguins. PO₂ and SO₂ were higher in the G_art group than in the other groups. Both venous and arterial blood samples may be used to evaluate the acid-base profile of Magellanic penguins.