To identify feasible indicators to evaluate animals’ emotional states as a parameter to assess animal welfare, the present study aimed at investigating the accuracy of free choice profiling (FCP) and fixed list (FL) approach of Qualitative Behaviour Assessment (QBA) in horses during the loading phase of transport. A total of 13 stakeholders were trained to score 2 different sets of videos of mixed breed horses loaded for road transport, using both FCP and FL, in 2 sessions. Generalized Procustes Analysis (GPA) consensus profile explained a higher percentage of variation (80.8%) than the mean of 1000 randomized profiles (41.2 ± 1.6%; p = 0.001) for the FCP method, showing an excellent inter-observer agreement. GPA identified two main factors, explaining 65.1% and 3.7% of the total variation. Factor 1 ranging from ‘anxious/ to ‘calm/relaxed’, described the valence of the horses’ emotional states. Factor 2, ranging from ‘bright’ to ‘assessing/withdrawn’, described the arousal. As for FL, Principal Component Analysis (PCA) first and second components (PC1 and PC2, respectively), explaining on average 59.8% and 12.6% of the data variability, had significant agreement between observers. PC1 ranges from relaxed/confident to anxious/frightened, while PC2 from alert/inquisitive to calm. Our study highlighted the need for the use of descriptors specifically selected, throughout a prior FCP process for the situation we want to evaluate to get a good QBA accuracy level.