Anion-π interactions are emerging as exotic features with potential applications in chemistry. In the last years, their relevance in living systems has been outlined, and so far there is no concluding significant evidence recognized about the participation of anion-π interactions in water because anion-π sensors contain large aromatic hydrophobic surfaces with limited solubility. By transforming a neutral heterocycle (for example quinoline) into its corresponding salt (quinolinium), we have been able to overcome these solubility issues, and new cationic water-soluble fluorophores have been prepared. Herein, we used N-alkylated heterocycles as π-acidic surfaces to shed light on the nature of anion-π in water by the direct measurement of the fluorescence and UV/Vis spectra in combination with DFT and X-ray analyses.