The antibiotic susceptibility of bacterial pathogens is typically determined based on planktonic cells, as recommended by several international guidelines. However, most of chronic infections – such as those established in wounds, cystic fibrosis lung, and onto indwelling devices – are associated to the formation of biofilms, communities of clustered bacteria attached onto a surface, abiotic or biotic, and embedded in an extracellular matrix produced by the bacteria and complexed with molecules from the host. Sessile microorganisms show significantly increased tolerance/resistance to antibiotics compared with planktonic counterparts. Consequently, antibiotic concentrations used in standard antimicrobial susceptibility tests, although effective against planktonic bacteria in vitro, are not predictive of the concentrations required to eradicate biofilm-related infections, thus leading to treatment failure, chronicization and removal of material in patients with indwelling medical devices.Meeting the need for the in vitro evaluation of biofilm susceptibility to antibiotics, here we reviewed several methods proposed in literature highlighting their advantages and limitations to guide scientists towards an appropriate choice.