This study aimed to develop a scientific and practical tool to be used to assess horse welfare after commercial transport over long journeys. A set of physical, behavioural and environmental measures was selected, covering welfare aspects of both transport and unloading procedures. The protocol was field-tested on 51 intra-EU commercial transports arriving at different sites in Italy. Univariate analysis was implemented to look for associations between the input variables (environmental hazards potentially affecting the animal well-being during long transports) and the outcome variables (direct evaluation of the animal condition). No severe welfare impairments were recorded (ie dead on arrival, severe injuries, non-ambulatory animals), while milder ones were more frequent at unloading (eg slipping; 36.7%, reluctance to move; 9.6%). Correlations emerged between ramp slope and falling; type of ramp floor and slipping; fast gait and the presence of gaps between the ramp and the floor. The horses' behaviour was also related to the type of handling procedure used. The measures were repeatable and practical to apply and score during real-time unloading. This work provides a sound basis for a new and practical welfare assessment tool for horses travelling over long journeys. Careful and constant application of this protocol would provide stakeholders with the opportunity to track and monitor changes in the industry over time, as well as to identify high risk areas in transport routines.