Cortisol levels reflect hypothalamic-pituitary-adrenocortical (HPA) axis activity. While most plasma cortisol is supposed to be bound to the soluble corticosteroid-binding globulin (CBG), only free cortisol (FC) actively regulates metabolic and immunological processes. We aimed to establish a multispecies suitable method to assess FC in cows and horses which in combination with total cortisol (TC) allows interpreting proportional changes of cortisol in saliva as well as in blood in response to a standardized HPA axis activation via ACTH. We further investigated if the ratios of cortisol fractions as obtained at basal levels in healthy horses (herbivorous and monogastric) and dairy cows (herbivorous and ruminant) change during HPA axis activation, and to which extent saliva cortisol (SC) is representative for alterations in plasma FC and adrenal cortex reactivity. However, it was not the objective of the present study to directly compare the two species. Dosages of ACTH applied in cows and horses were based on published data. Synthetic ACTH was intravenously administered to eight dairy cows (0.16 µg/kg BW) and five horses (1 µg/kg BW). Blood and saliva were collected every 30 min for 3 h from a jugular vein catheter, and analyzed for TC and SC, the ratio of free cortisol (rFC), and the concentration of FC (cFC) in plasma. During the entire sampling period of the ACTH test, plasma TC was paralleled by blood cFC, rFC, and SC in both cows and horses. All cortisol fractions increased within 30 min of ACTH administration compared to basal values (0 min, P < 0.05). Peak TC concentration reached 63.2 ± 9.6 ng/mL and 73.2 ± 11.8 ng/mL in bovine and equine plasma, respectively. Peak values of rFC averaged 17.9 ± 4.5% in cows and 19.2 ± 7.8% in horses. The ratio of SC to cFC in horses remained similar during the ACTH challenge suggesting that SC is recruited from plasma FC. However, SC increased less compared to plasma TC and FC during HPA axis activation in cows. In conclusion, the short-term activation of the HPA axis caused not only an elevation of TC, but also a similar increase of rFC in both species. SC closely reflected changes of FC in horses, but less accurately in cows. The concomitant evaluation of changes among cortisol fractions might give further indications on adaptation mechanisms in glucocorticoid regulation as well as differentiate cortisol-related health disorders.