Endocrine diagnostics currently depend on the ability to measure low and high concentrations of diagnostic hormones using immunoassays. This often is challenging in species other than humans, dogs, cats, and horses due to lack of validated assays and reference intervals. There are strategies to approach endocrine testing in zoo, wildlife, and zoologic companion animals but caution is needed in interpreting results. Newer techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) may be more useful for all species, although technical hurdles remain for this method too.