Plasmin is a protease in milk, which concentration is increased during mastitis. The aim of the study was design and characterize short, tailor made fluorogenic substrates for plasmin to see differences in plasmin activity in healthy milk compared to mastitic milk. According to the specificity matrix of plasmin, containing 125 currently known plasmin substrates, six novel plasmin specific substrates were designed and characterized. Eight other substrates were insensitive for plasmin, which was in line with our expectations. The plasmin-sensitive substrates showed no cross-reactivity with proteases from mastitogenic bacteria in vitro. A proof of concept to distinguish healthy milk from mastitic milk is described. Potentially, these substrates present a rapid alternative to the currently established methodologies to detect mastitis in milk.