The success of nontargeted analysis often depends on libraries containing reference mass spectra of known chemical compounds; the mass spectra of unknown compounds are compared to these reference mass spectra, leading to a probable compound identity. Typical calculations include the mean measured values for each ion m/z and intensity with no estimation of the variability of the measurement. This study presents a novel tool for the calculation of the variability of a measured mass spectrum, including the various data parameters that can impact the measured variability. Using perfluorooctanoic acid (PFOA) as the model compound, the variability of measured data-dependent fragmentation mass spectra (ddMS2) was calculated within replicate measurements of a simple solution of PFOA and a complex mixture (house dust extract) containing PFOA. The variability of the measured ddMS2 for PFOA in the solution and house dust extract were similar, with standard deviations about the measured m/z value ranging from m/z 0.00003 to 0.00015 and the standard deviations about the measured relative intensity ranging from 0.0077 to 0.0211 relative intensity units. In addition, the selected parameters for the extraction of ddMS2 from a single analytical run varied between the sample types due to the increased presence of background ions in the house dust extract. Finally, the variability of the ddMS2 spectra for PFOA in both samples was used to calculate a more robust similarity factor, informing the confidence of the identification of unknown compounds.