Electrophoresis is a useful diagnostic tool for detecting inflammation, including inflammation associated with infectious diseases (eg, aspergillosis in penguins). To our knowledge, reference intervals are not available for plasma proteins via electrophoresis in Humboldt penguins (Spheniscus humboldti). Therefore, preliminary reference intervals for blood plasma proteins measured by capillary zone electrophoresis were calculated for Humboldt penguins from a single zoological collection, and possible differences between the sexes and the ages of the birds were evaluated. Lithium heparinized plasma samples from 39 Humboldt penguins were analyzed. The following sex- and age-independent reference intervals were calculated: total protein 33.8–70.4 g/L, prealbumin 1.9–4.9 g/L, albumin 12.9–31.1 g/L, albumin: globulin ratio 0.7–1.7, α-globulins 4.5–11.6 g/L, β-globulins 5.6–20.6 g/L, and γ-globulins 2.6–8.4 g/L. Male penguins had a significantly (P = 0.047) higher albumin: globulin ratio and lower percentage of β-globulins (P = 0.015) in comparison with female penguins. Prealbumin (g/L) significantly (P = 0.021) decreased with increased age of the penguins. These results showed some differences between the sexes and ages of the penguins, which should be considered when interpreting the results. Further studies are needed to determine whether differences in other age groups or seasons exist, and also to evaluate which infectious diseases affect plasma proteins and how the reference values calculated here may deviate in ill penguins.