Enterobacter species are classified as high-priority pathogens due to high prevalence of multidrug resistance from persistent antibiotic use. For Enterobacter infections caused by multidrug-resistant isolates, colistin (polymyxin E), a last-resort antibiotic, is a potential treatment option. Treatment with colistin has been shown to lead to emergence of polymyxin resistance. The primary mechanism for colistin resistance is modification of terminal phosphate moieties of lipid A, leading to decreased membrane electronegativity and reducing colistin binding affinity. Detection of these modifications, including the addition of phosphoethanolamine and 4-amino-4-deoxy-l-arabinose (Ara4N), can be used for prediction of colistin resistance using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The objective of this study was to identify lipid A markers for colistin resistance in Enterobacter species and Klebsiella aerogenes (formerly Enterobacter aerogenes). Using a collection of Enterobacter and Klebsiella aerogenes clinical isolates, broth MICs for colistin were determined initially. Subsequently, killing assays were carried out to determine how the concentration of colistin at which there is approximately 50% survival (kill50) equates to their MICs. Finally, lipid A analysis was conducted via MALDI-TOF MS using the novel rapid extraction method, termed fast lipid analysis technique (FLAT), to correlate MIC and killing efficacy with predictive lipid A modifications. Sensitivity and specificity of the MS assay compared to MIC interpretation were 100% and 53.4%, respectively. A receiver operator characteristic (ROC) demonstrated that MS was highly correlated with killing, with area under the curve of 0.97. This analysis demonstrated the potential utility of MALDI-TOF MS as a rapid diagnostic platform of colistin resistance in Enterobacter species. IMPORTANCE In this study, we develop a novel method for identifying colistin resistance in Enterobacter species and Klebsiella aerogenes without performing antimicrobial susceptibility testing. Typically, susceptibility testing requires an additional 24 to 48 h, while the MS assay described in this study allows for resistant identifications in under 1 h after initial culture. Identification using MALDI-TOF MS would save time and prevent inappropriate use of colistin. MALDI-TOF MS is an easy-to-use, readily available, robust diagnostic tool in clinical laboratories. Furthermore, this study highlights limitations of polymyxin susceptibility testing. Use of a killing assay best captures how colistin treats infection and is shown to be highly correlated with our MS assay; thus, the MS assay in this study effectively predicts how colistin would treat a patient's infection. Use of MALDI-TOF MS for accurate and early identification of antimicrobial resistance can improve antimicrobial stewardship and patient outcomes.