Because clinical mastitis, one of the most common diseases in dairy cows, is routinely treated with antimicrobial substances, it offers a high potential for future reduction of antimicrobial usage. In fact, intramammary antibiotic administration is not advisable in cases of clinical mastitis caused by coliform bacteria, yeasts, or protothecae or in cases with no detectable mastitis pathogen. To avoid unnecessary treatments with antimicrobials for the benefit of animal health and public welfare, the rapid identification of the mastitis-causing pathogens becomes necessary. Therefore, 4 different incubation time schemes for a newly developed tube test system (MastDecide, Quidee GmbH, Homberg, Germany) were analyzed in terms of sensitivity, specificity, negative and positive predictive values, and apparent and true prevalence compared with the conventional microbiological investigation results for 251 clinical mastitis milk samples from 11 dairy farms located in northern Germany. An aliquot (100 µL) of a quarter foremilk sample was taken in both cases. The evaluation of the tube test result after 14 h of incubation at 37°C resulted in sensitivity values of 83.6, 72.2, and 70.7% and specificity values of 94.1, 83.3, and 90.8% for gram-positive cocci, coliform bacteria, and no growth or further pathogens, respectively. Moreover, for the present pathogen distribution, the overall tube test sensitivity was highest after 14 h of incubation (sensitivity = 80.9%; specificity = 70.7%). The described tube test system has the potential to provide a new option for an evidence-based mastitis therapy, with the aim of reducing the future usage of antimicrobials in dairy cows and a larger goal of decreasing antimicrobial resistance. However, a subsequent on-farm test validation should be performed before implementation in an evidence-based mastitis therapy concept can be recommended.