Antibiotic susceptibility testing (AST) by agar diffusion has been repeatedly standardized and, in most cases, gives results which predict clinical success when antibiotic treatment is based on such results. The formation of the inhibition zone is due to a transition from planktonic to biofilm mode of growth. The kinetics of the interaction of antibiotics with bacteria is similar during AST by agar diffusion and during administration of antibiotics to the patients. However, the Mueller‐Hinton agar (MHA) recommended for AST agar diffusion test is fundamentally different from the composition of the interstitial fluid in the human body where the infections take place and human cells do not thrive in MH media. Use of RPMI 1640 medium designed for growth of eucaryotic cells for AST of Pseudomonas aeruginosa against azithromycin results in lower minimal inhibitory concentration, compared to results obtained by MHA. The reason is that the RPMI 1640 medium increases uptake and reduces efflux of azithromycin compared to MHA. During treatment of cystic fibrosis patients with azithromycin, mutational resistance occur which is not detected by AST with MHA. Whether this is the case with other antibiotics and bacteria is not known but it is of clinical importance to be studied.