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Stress exposure during perinatal period may lead to maternal cortisol increase that negatively affects the offspring development. In recent years, the interest on non-invasive sampling methods to measure cortisol as a marker of stress is increasing in both humans and animals. Indeed, discomfort due to blood collection may compromise the diagnostic outcome, mainly in uncooperative patients. So far, some alternative matrices but not milk have been explored in adult dogs, while no data are available on the neonate and paediatric live pups. This study aimed to measure cortisol concentration in different biological substrates in both dams (blood, saliva, hair and milk) and pups (saliva and hair) at established times from proestrus up to two months after parturition. For this purpose, five female German shepherd bitches and their 22 pups were enrolled. Cortisol concentration was assessed using the enzyme immunoassay kit (Salivary Cortisol ELISA kit, Salimetrics) after matrices appropriate preparation if required. Cortisol was measurable in all the substrates, except some milk samples below the detection limit. Maternal cortisol concentrations differed among the matrices (P <0.0001) with the highest values recorded in plasma (median 0.596 μg/dL) compared to saliva (median 0.159 μg/dL), hair (median 0.083 μg/dL) and milk (median 0.045 μg/dL). Cortisol in dams did not vary within the same matrix over time. In pups, salivary (median 0.295 μg/dL) cortisol was always higher than hair (median 0.049 μg/dL; P <0.0001). At birth (P = 0.01) and two months later (P = 0.05), neonatal salivary cortisol was higher compared to other samplings. The present study demonstrates the suitability of these innovative substrates for cortisol measurement, suggesting them as potential diagnostic support in canine neonatology and welfare., Competing Interests: The authors have declared that no competing interests exist.