The pre-slaughter period includes a series of essential steps (loading, transport, unloading, lairage, and slaughter) likely to induce physical and/or psychological stress in cattle. Blood cortisol concentration is a reliable indicator of stress, particularly psychological, as it reacts more slowly to stress factors and recovers less quickly than other markers. This study aims to evaluate the effects of animal-related, environmental, and slaughter procedure factors on plasma cortisol levels in cattle at the time of bleeding. A total of 160 cattle of different breeds and ages were observed. Collected data included origin (farm or market), breed, age, transport duration, lairage time, and waiting time in the slaughter room. Blood samples were collected at bleeding, and Cortisol levels were measured using the VIDAS automated system, which uses the ELFA (Enzyme-Linked Fluorescent Assay) method, combining ELISA (Enzyme-Linked Immunosorbent Assay) with final fluorescence reading in blue light. The mean concentration observed was 35.26 ng/mL. Significant differences were noted between breeds, lairage durations, and waiting times in the slaughter room. However, age, origin, and season did not show significant differences. The results indicate variability in stress reactivity depending on the breed.