Antibiotic susceptibility of clinical mastitis pathogens has traditionally been determined using the agar diffusion method that was designed to reflect the antibiotic concentration in serum and interstitial fluid of human patients after receiving oral or intravenous administration. The validity of applying agar diffusion susceptibility breakpoints derived from humans to the treatment of bovine mastitis has not been established and is extremely questionable because (1) bovine milk pH and electrolyte, fat, protein, and leukocyte concentrations, growth factor composition, and pharmacokinetic profiles are different than those for human plasma and (2) human bacterial pathogens are often different from bovine mastitis pathogens. Also, antibiotics are distributed unevenly in an inflamed gland, and high antibiotic concentrations can alter neutrophil morphology or function in vitro and thereby inhibit bacterial clearance in vivo. The current cost of antibiotic susceptibility testing is $12 to $20 per test. Because the dairy industry is economically driven, any diagnostic test should be validated, have appropriate sensitivity and specificity, and have an acceptable economic return on the cost of testing before it can be routinely recommended. In the authors' opinion, antimicrobial susceptibility testing of mastitis pathogens has not been adequately validated for most mastitis pathogens and antibiotics; therefore, the authors do not currently recommend the use of susceptibility testing to guide treatment decisions for individual cows. Additional research is needed to further define the role, if any, that antimicrobial susceptibility testing should play in the treatment of clinical mastitis.