Cortisol and corticosterone, hormones traditionally considered biomarkers of stress, can be measured in fluid biomatrices (e.g., blood, saliva) from live animals to evaluate conditions at sampling time, or in solid biomatrices (e.g., hair, feather) from live or dead animals to obtain information regarding long-term changes. Using these biomarkers to evaluate physiological stress responses in domestic animals may be challenging due to the diverse characteristics of biomatrices for potential measurement. Ideally, a single measurement from the biomatrix should be sufficient for evaluating chronic stress. The availability of appropriate and cost-effective immunoassay methods for detecting the biomarkers should also be considered. This review discusses the strengths and limitations of different biomatrices with regard to ensuring the highest possible reliability for chronic stress evaluation. Overall, solid biomatrices require less frequent sampling than other biomatrices, resulting in greater time- and cost-effectiveness, greater ease of use, and fewer errors. The multiplex immunoassay can be used to analyze interactions and correlations between cortisol and other stress biomarkers in the same biomatrix. In light of the lack of information regarding appropriate biomatrices for measuring chronic stress, this review may help investigators set experimental conditions or design biological research.