Australian livestock industries face increased scrutiny from animal welfare groups and society, and the long-distance transport of livestock by sea has recently gained particular attention. Other than non-compliance with broad regulatory standards and voyage mortality rates, there is minimal information to ascertain the welfare of exported livestock. There is currently no standardised, validated animal welfare assessment protocol for livestock on-farm prior to live export or when undergoing transport. This study describes a novel assessment protocol suitable for use on live feeder and slaughter animals exported by sea from Australia. Health and welfare indicators for use in the livestock export supply chain were identified by reviewing three internationally recognised animal welfare assessment protocols for livestock; Welfare Quality®, AWIN and AssureWel, as well as consulting with industry compliance standards and guidelines. This paper proposes a welfare protocol designed to assess sheep and beef cattle exported by sea from Australia, and incorporates environmental-, resource-, management- and animal-based measures. In collaboration with industry, this welfare protocol can be tested on commercial livestock consignments, and be used for ongoing management, for increased transparency and to provide feedback to operators for continuous improvement.