Alkaline folates self-associate in aqueous solutions to form columnar lyotropic phases. Such phases are made by quadruplexes, which are supramolecular helicoidal structures formed by a stacked array of folate tetramers. High-pressure synchrotron X-ray diffraction is used to analyze alkaline folate quadruplex stability and energetics. Diffraction data show that both inter-helical lateral and tetramer stacking distances decrease as a function of pressure. Lateral and axial quadruplex compressibilities and force constants have been derived and strong correlation between the strength of tetramer stacking and pressure effects demonstrated. In particular, quadruplex rigidity increases by changing Na+ to K+ and by adding excess KCl, as a consequence of increased stacking interactions and quadruplex elongation.