Conventional antibiotic susceptibility testing (AST) assays such as broth microdilution and Kirby-Bauer disk diffusion are time-consuming (e.g., 24-72 h) and labor-intensive. Here, we present a microfluidic platform to perform AST assays with a broad range of antibiotic concentrations and controls. A culture medium stream was serially enriched with antibiotics along the length of the platform via diffusion and flow-directing mass convection mechanisms, generating a concentration gradient captured in a series of microchamber duplicates. We observed an agreement between the simulated and experimental concentration gradients and applicability to a variety of different molecules by changing the loading time according to a simple linear equation. The AST assay in our platform is based on bacterial metabolism, indicated by resazurin fluorescence. The small reaction volume enabled a minimum inhibitory concentration (MIC) to be determined in 4-5 h. Proof-of-concept functionality testing, using human isolates and clinically important antibiotics from different classes, indicated a high rate of agreement (94%: MIC within ±1 two-fold dilution of the reference method) of on-chip MICs and conventional broth microdilution. Overall, our results showed that this microfluidic platform is capable of determining antibiotic susceptibility in a rapid and reliable manner.