The worldwide impacts of infectious diseases drive the development of novel diagnostics and therapeutics to combat them. Rapid and accurate diagnoses of bacterial infections enable clinicians to quickly initiate targeted antimicrobial chemotherapy, reducing patient morbidity and mortality. The advent of a mass spectrometry-based platform targeting bacterial proteins represents a new and promising technology that simplifies the clinical workflow and reduces time and cost from conventional methodologies. The approach makes identifications via unique protein mass spectra by comparison against a reference database of spectra from known sources. A wealth of research regarding essential glycolipids, which are found in abundance in microbial membranes, has revealed tremendous structural diversity among these molecules that is species- and often intra-species-specific in response to environmental signals. This thesis introduces a novel diagnostic platform that takes advantage of this specificity to identify the ESKAPE pathogens via their unique glycolipid mass spectral fingerprint in a manner similar to MS protein phenotyping. Importantly, differentiation on the basis of colistin susceptibility is demonstrated in select Gram-negative bacteria and conferred by the recently discovered plasmid-mediated mcr-1 gene. The platform also detects and identifies organisms directly from patient specimens illustrating the potential for this platform to be culture-free. These capabilities offer significant advantages over protein typing, and the glycolipid library exists on the same instrumentation suggesting that the two approaches could complement each other offering an additively more powerful diagnostic platform.