Instrumentation for mass spectrometry has undergone continuous improvements in terms of hardware and software. Methods for ionization have evolved from atmospheric pressure ionization (e.g., electron ionization, atmospheric pressure chemical and photochemical ionization) to matrix-assisted laser desorption ionization, and more recently, to ambient ionization, which enable direct injection of samples obviating the need for prior chromatographic separations of complex mixtures. There have been significant developments in the manner by which mass spectrometers filter ions. In addition to quadrupole (Q) and ion traps which detect integer mass-to-charge ratios, time-of-flight (TOF) and orbitrap enable high resolution mass detection to enable exact molecular formula detection. Combining multiple filtering elements such as triple quadrupole (QQQ) enable ion fragmentation for structural identification, while quadrupole-TOF (Q-TOF) enable enhanced analytical performance. With hardware improvements, different data acquisition methods have evolved to include full scan, product and precursor ion scan, neutral loss scan and selected reaction monitoring for QQQ, and data dependent and independent acquisitions for Q-TOF. The next generation of mass spectrometers are headed into diverse directions: integration with total clinical laboratory automation, point-of-care testing for real-time intraoperative detection of key analytes, and artificial intelligence for enhanced data analysis.