White spot lesions occur when the pathogenic bacteria have broken through the enamel layer. White spot lesions (WSLs) can be treated using a complex approach. The most crucial step is to stop demineralization and biofilm formation and use assorted strategies for remineralization of lesions, thinning, microabrasion, erosion infiltration, adhesive composite resin restorations, and the bonded facets. To evaluate and compare the fluoride, calcium, and phosphorus ion release, remineralizing efficacy, and microhardness of zwitterionic material, self-assembling peptide, and bioactive glass (BGA) incorporated with MI Varnish. The original study was conducted on 60 extracted premolars; the sample size calculated was 10 per group. All samples were divided into four groups-group A, MI Varnish (control), group B, MI Varnish + zwitterionic material, group C, MI Varnish + self-assembling peptide, and group D, MI Varnish + BGA. All these groups were further evaluated and compared for fluoride, calcium, and phorphorus ion release, remineralizing efficacy, and surface microhardness (SMH). Zwitterionic material, when incorporated with MI Varnish showed high fluoride and calcium ion release and high remineralizing efficacy under polarized light microscopy (PLM). BGA, when incorporated with MI Varnish showed high phosphorus ion release and higher values in the evaluation of SMH, followed by zwitterionic material and self-assembling peptide. MI varnish alone had remineralizing properties of WSLs, but when novel materials like zwitterionic ion, self-assembling peptide, and BGA are incorporated, its efficacy increases. Among all zwitterionic ions showed superior results for fluoride and calcium ion release and remineralization and BGA for phosphorus ion release and SMH. Patil SV, Gugwad SC, Devendrappa SN, et al. Comparative Evaluation of Zwitterionic Material, Self-assembling Peptide, and Bioactive Glass Incorporated with MI Varnish for Fluoride, Calcium, and Phosphorus Ion Release, Enamel Remineralization, and Microhardness. Int J Clin Pediatr Dent 2024;17(S-1):S37-S42.