Gram-negative bacteria (GNBs) are common pathogens causing severe sepsis. Rapid evaluation of drug susceptibility would guide effective antibiotic treatment and promote life-saving. A total of 78 clinical isolates of 13 Gram-negative species collected between April 2013 and November 2013 from two medical centers in Tainan were tested. Bacterial morphology changes in different concentrations of antibiotics were observed under the electric field of a quadruple electrode array using light microscopy. The minimal inhibitory concentrations (MICs) of four antimicrobial agents, namely, cefazolin, ceftazidime, cefepime, and doripenem, were determined by the dielectrophoretic antimicrobial susceptibility testing (dAST) and by the conventional broth dilution testing (BDT). The antibiotics at the concentration of 1× MIC induced obvious morphological changes in susceptible GNBs, including cell elongation, cell swelling, or lysis, at 90 min. In contrast, resistant strains remained unchanged. The MIC results measured by dAST were in good agreement with those of BDT (essential agreement 95.6%). The category agreement rate was 89.2%, and the very major errors rate for dAST was 2.9%. In conclusion, dAST could accurately determine drug susceptibility within 90 min. Comprehensive tests by dAST for more drugs against more GNB species are possible in the future.