A new separation-based analytical method was developed to evaluate the multivalency and cooperativity of supramolecular host-guest complexation in porous materials. The method is based on inverse mixed-mode chromatography in which a porous material with a multivalent functional group is packed into a column and bound with a complementary guest molecule to form a multivalent complex. The bound guest molecules are eluted in the mobile phase and detected by appropriate methods such as UV absorption. The retention factor of the guest molecule is determined and broken down into the contributions of noncovalent interactions between binding sites (e.g., hydrophobic and ionic components), thereby calculating the effective molarity and cooperativity factor of the complexation. Two model systems denoted as RP/SCX and RP/SAX were analyzed by the established method. On average, the RP/SCX system has an effective molarity (EM) of 0.14 M and a cooperativity factor (beta) of 0.86, while the RP/SAX system has an EM value of 0.18 M and a beta value of 2.3. Interestingly, experiments have shown that these values do not change with changes in the intrinsic binding strength of the constituent sites. In summary, the developed method allows for quantitative assessment of multivalency and cooperativity effects in porous materials, providing a valuable complement to the analytical toolbox for supramolecular chemists and materials scientists.