Transportation duration and distance are significant concerns for animal welfare, particularly in the poultry industry. However, limited proteomic studies have investigated the impact of transport duration on poultry welfare. In this study, mass spectrometry based bottom up proteomics was employed to sensitively and impartially profile the liver tissue proteome of chickens, addressing the issue of animal stress and welfare in response to transportation before slaughter. The liver exudates obtained from Ross 508 chickens exposed to either short or long road transportation underwent quantitative label-free LC-MS proteomic profiling. This method identified a total of 1,368 proteins, among which 35 were found to be significantly different (p < 0.05) and capable of distinguishing between short and long road transportation conditions. Specifically, 23 proteins exhibited up-regulation in the non stressed group, while 12 proteins showed up-regulation in the stressed group. The proteins identified in this pilot study encompassed those linked to homeostasis and cellular energetic balance, including heat shock proteins and the 5'-nucleotidase domain-containing family. These results contribute to a deeper understanding of the proteome in broiler liver tissues, shedding light on poultry adaptability to transport stress. Furthermore, the identified proteins present potential as biomarkers, suggesting promising approaches to enhance poultry care and management within the industry.