In this work, we develop a mathematical framework for quantifying the relative abundance of di-Ub isomers in complex mixtures using IM-MS. We found that despite the structural similarity, different di-Ub isomers could have significantly different response factors that do not allow linear approximation to convert IM intensities into molar fractions of a particular isomer. Using an internal standard and applying multiple linear regression analysis to the spectral data enables robust quantitation of di-Ub molar fractions in complex mixtures containing up to four different isomers. Our mathematical model can also be used to characterize the types of di-Ub isomers produced by enzymatic reactions. Future efforts will focus on using these methods to characterize mixtures of higher molecular weight Ub conjugates with greater morphological complexity.