This paper focuses on the different methods of cortisol assessment, highlighting the increasing use of non-invasive methods for cortisol assessment. After reviewing over 18,000 studies, 78 relevant ones were identified, clearly showing a shift towards less harmful methods. The ethics committee recommends that research methods remain as non-invasive as possible, stressing the importance of minimalizing discomfort for research animals. Invasive methods may compromise animals’ welfare and influence physiological parameters. The employment of non-invasive methods of sampling could enhance the reliability of the results by avoiding confounding effects related to stress-induced hormonal changes. Many factors, including environmental, genetic, and social ones, influence cortisol levels. Challenges such as small sample sizes and inconsistent protocols are still a major concern. This study calls for interdisciplinary research combining behavioural and physiological measures, which will help refine different cortisol measurement techniques. Future findings may improve major concerns regarding canines’ welfare and ensure ethical research practices.