Specific interactions among biomolecules to form noncovalently bound complexes play a pivotal role in key cellular processes such as cell division, cell signalling, gene transcription and translation. The propensity of noncovalently bound complexes to dissociate into their components can be quantified and constants of dissociation (K_d) can be obtained by various methods. Mass spectrometry has become an important method to measure K_d. The advent of soft ionisation techniques, in particular electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI) has established mass spectrometry as a viable technique for investigating noncovalent interactions and for quantifying their binding strengths. Under carefully chosen experimental and instrumental conditions, it is possible to observe intact noncovalent complexes in the gas phase using ESI and MALDI, and to use the mass spectra as a read-out for determining solution-phase K_d. Compared to other biophysical methods, mass spectrometry is highly sensitive and fast, and gives additional information about the stoichiometry and specificity of noncovalent interactions. This review focuses on recent MS-based methodologies for quantification of binding strengths, in particular those that promise to complement conventional biophysical methods.