AP-MALDI MS profiling coupled with multivariate analysis for the detection of farm-related diseases using a simple one-pot sample preparation of milk. Milk samples were collected at the Centre for Dairy Research (CEDAR) of University of Reading (UK). The analysis performed on the test set allowed the detection of clinical mastitis with a correct classification rate of 98.57% and the correct detection of mastitis 2 days before the clinical event with a sensitivity of 70% and a specificity of 100%.