Mobilized colistin resistance (mcr) genes confer resistance to colistin, a last-resort antibiotic for multidrug-resistant Gram-negative infections. In this case report, we describe a novel lipid-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) diagnostic used to rapidly identify an mcr-1-positive Escherichia coli directly from a patient with a urinary tract infection without the need for ex vivo growth.