Over the past decade, matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry has revolutionized the practice of clinical microbiology and infectious disease diagnostics. Rapid advancement has occurred through the development and implementation of mass spectrometric protein profiling technologies that are widely available. Ease of sample preparation, rapid turnaround times, and high throughput accuracy have accelerated acceptance within the clinical laboratory. New mass spectrometric technologies centered on multiple microbial diagnostic markers are in development. Such new applications, reviewed in this article and on the near horizon, stand to greatly enhance the capabilities and utility for improved mass spectrometric microbial identification and patient care.