Carbapenemase producing Enterobacteriaceae (CPE) are becoming a global healthcare concern. Current laboratory methods for the detection of CPE include screening followed by confirmatory phenotypic and genotypic tests. These processes would generally take >\/= 72 hours, which could negatively impact patient care and Infection Control practices. To this end, we developed a protocol for rapid resistance testing (RRT) to detect hydrolysis in a panel of beta lactam antibiotics consisiting of ampicillin, cefazolin, cefotaxime and imipenem, using liquid chromatography tandem mass spectrometry. Ninety-nine beta lactamase producing Enterobacteriaceae isolates were used to evaluate the RRT method, 54 isolates were CPE and 45 isolates were Class A or AmpC beta lactamase producing Enterobacteriaceae but not carbapenemase producers. We also tested 10 E.coli isolates that were susceptible to ampicillin, cefazolin, cefotaxime and imipenem. Receiver Operating Characteristic (ROC) Curves analysis showed that imipenem had a sensitivity and a specificity of 100% for carbapenemase detection at hydrolysis cut off values that are greater than 50% and less than or equal to 80%. The RRT protocol can be conducted in a time frame of less than 2 hours. This preliminary study shows that the rapid resistance testing protocol might have utility for the rapid detection of CPE. Additional work with a greater number and a variety of beta-lactamase producing Enterobacteriaceae isolates is required to validate these preliminary findings.