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Antimicrobial susceptibility testing (AST) is a key measure in clinical microbiology laboratories to enable appropriate antimicrobial administration. During an AST, the determination of the minimum inhibitory concentration (MIC) is an important step in which the bacterial responses to an antibiotic at a series of concentrations obtained in separate bacterial growth chambers or sites are compared. However, the preparation of different antibiotic concentrations is time-consuming and labor-intensive. In this paper, we present a microfluidic device that generates a concentration gradient for antibiotics that is produced by diffusion in the laminar flow regime along a series of lateral microwells to encapsulate bacteria for antibiotic treatment. All the AST preparation steps (including bacterium loading, antibiotic concentration generation, buffer washing, and isolated bacterial growth with an antibiotic) can be performed in a single chip. The viable bacterial cells in each microwell after the antibiotic treatment are then quantified by their surface-enhanced Raman scattering (SERS) signals that are acquired after placing a uniform SERS-active substrate in contact with all the microwells. For proof-of-concept, we demonstrated the AST performance of this system on ampicillin (AMP)-susceptible and -resistant E. coli strains. Compared with the parameters for conventional AST methods, the AST procedure based on this chip requires only 20 μL of bacteria solution and 5 h of operation time. This result indicates that this integrated system can greatly shorten and simplify the tedious and labor-intensive procedures required for current standard AST methods.