Animal testing for toxicity assessment of chemicals and pharmaceuticals must take the 3R principles into consideration. During toxicity testing in vivo, clinical signs are used to monitor animal welfare and to inform about potential toxicity. This study investigated possible associations between clinical signs, body weight change and histopathological findings observed after necropsy. It was hypothesized that clinical signs and body weight loss observed during experiments could be used as early markers of organ toxicity. This represents a potential for Refinement in terms of improved study management and decrease of pain and distress experienced during animal experiments. To this end, data from three sequential toxicity studies in rats were analyzed using the multivariate partial least squares (PLS) regression method. Associations with correct prediction over 80% were found between the occurrence of mild to severe clinical signs and histopathological findings in the thymus, testes, epididymides and bone marrow. Piloerection, eyes half shut and slightly decreased motor activity showed the strongest associations to the pathological findings. A 5% body weight loss was found to be a strong empirical predictor of pathological findings but could also be predicted accurately by clinical signs. Thus, we suggest using mild clinical signs and a 5% body weight loss as toxicity markers, and as a non-invasive surveillance tool to monitor research animal's welfare and toxicity testing. These clinical signs may also enable Reduced animal use due to their informative potential to support scientific decisions regarding drug candidate selection, dose setting, study design and toxicity assessment.