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This current software is capable of deconvoluting heterogeneous, complex, and noisy native mass spectra of large protein assemblies as demonstrated by analysis of (a) synthetic mononucleosomes containing severely overlapping peaks, (b) an RNA polymerase II/a-amanitin complex with many closely interleaved ion signals, and (c) human TriC complex containing high levels of background noise. Charge state distributions of the molecular species are determined by fitting linear combinations of charge envelopes to the overall experimental mass spectrum. Overlapped Peaks are detected by examination of the second derivative of the raw mass spectrum. In this report we describe a comprehensive algorithm developed for addressing peak detection, peak overlap, and charge state assignment in native mass spectra, called PeakSeeker. This situation presents a challenge for peak detection, correct charge state and charge envelope assignment, and ultimately extracting the relevant underlying mass values of the non-covalent assemblages being investigated. The wide peak widths together with the fact that sequential charge state series from highly charged ions are closely spaced means that native spectra containing multiple species often suffer from high degrees of peak overlap or else contain highly interleaved charge envelopes. However, native spectra derived from these assemblies are often partially obscured by low signal-to-noise as well as broad peak shapes due to residual solvation and adduction after the electrospray process. Native electrospray-ionization mass spectrometry (native MS) measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact protein assemblies.