Here, we describe a mathematical approach to quantifying mixtures of Ub dimers using IM-MS. Calculating the molar fraction of individual chain linkage types in complex mixtures is challenging because the response to changes in fractional composition is non-linear. Using a multiple linear regression analysis approach, we are able to overcome these issues, and mixtures comprising at least four different isomers can be quantified. To assess the utility of our approach we measured the relative abundance of linkages produced during in vitro Ub chain assembly reactions and compared the results to those obtained by bottom-up AQUA analysis. We found that the molar fraction measured by IM-MS is consistent with AQUA data. Our study thus establishes a foundation for quantifying the relative abundance of different Ub chain linkage types using IM-MS and multiple linear regression analysis.