I-motifs are intercalated cytosine quadruplexes discovered in 1993, but they weren’t shown to exist in the cell nucleus until 2018. While their biological role has yet to be fully elucidated, they have been implicated in regulating expression of several genes involved in cancer, depression, diabetes, neurodegenerative diseases, and cardiovascular disease. The effect of monovalent cations on i-motif formation has been the subject of numerous reports with inconsistent results. The most notable of these discrepancies are contradictory reports of sodium and lithium ions stabilizing or destabilizing i-motifs. These inconsistencies could be due to the use of different buffer salts in these studies. The RAD17 promoter sequence has been reported to form i-motif structures at physiological pH and has been used as a model system to study potential interactions of buffer systems with DNA. Confirmation of i-motif formation was demonstrated with circular dichroism (CD) which can distinguish between various DNA secondary structures due to unique spectral signatures associated with the orientation of the nucleotide bases in solution. Small Angle X-Ray Scattering (SAXS) was utilized to provide additional information to validate structural assumptions from CD. Despite previous studies touting the stability of RAD17 as an i-motif, RAD17 only appeared to have formed i-motifs in HEPES buffer at pH 7.4, but not in buffers containing Tris at the same pH. Herein we demonstrated that differences in metal ion concentrations induce different structural changes to RAD17 depending on buffer selection.