The ability of Pseudomonas aeruginosa to form biofilm during a long-term infection makes it difficult to treat patients correctly. The current clinical antimicrobial susceptibility testing methods are based on the study of planktonic strains. A standardized protocol to analyze the antimicrobial susceptibility in biofilms is necessary for routine laboratories. The aims of this study were to develop a simple biofilm model and to study the antimicrobial susceptibility of P. aeruginosa strains in biofilm growth. Different artificial sputum media, and aerobiosis and microaerobiosis conditions were analyzed using a microtiter plate method and P. aeruginosa PAO1 as reference strain. Planktonic and biofilm antimicrobial susceptibility to cefepime, imipenem, azithromycin, gentamicin, tobramycin, and ciprofloxacin were determined in clinical and non-clinical P. aeruginosa strains. The Synthetic Cystic Fibrosis Medium was proposed as a good medium. The biofilm greatly increased the resistance to tested antimicrobials, except for azithromycin. Cefepime and imipenem showed poor anti-biofilm effect while tobramycin, gentamicin, and ciprofloxacin showed good activity in some strains. Azithromycin showed a better activity in biofilm than in planktonic state when aerobic conditions were used. This study establishes useful information to test antimicrobial susceptibility in P. aeruginosa biofilms, and includes possible antimicrobial options to treat long-term infected patients.