Keratoconus is a progressive disease characterized by corneal thinning and conical deformation. Corneal cross-linking, a common treatment, strengthens collagen fibers using vitamin B2 (riboflavin) and UVA light. However, the surgical removal of the corneal epithelium (Epi-Off) required for riboflavin penetration causes complications and may affect treatment success. To address this, research has focused on delivering riboflavin without removing the epithelium (Epi-On). In this study, riboflavin-based hydrogel and co-crystal formulations were developed and evaluated through in vitro, ex vivo, and in vivo studies. Co-crystals were prepared using trehalose, dextrose, mannitol, and nicotinamide as agents, employing solvent evaporation and co-mixing methods. These formulations were characterized using DSC (Differential Scanning Calorimetry), XRD (X-Ray Diffraction), and FTIR (Fourier Transform Infrared Spectroscopy), identifying 1R1N (1 unit mol riboflavin and 1 unit mol nicotinamide co-crystals) and 1R1M (1 unit mol riboflavin and 1 unit mol mannitol co-crystals) groups as promising candidates. Thermosensitive hydrophilic gels containing riboflavin or riboflavin-5-phosphate sodium and Transcutol P (a permeation enhancer) were also developed, using Pluronic F-127 as the polymer. The 18 % Pluronic F-127 gel formed at 31.4 ± 0.2 °C. Drug release studies showed faster release from riboflavin-5-phosphate sodium formulations, while ex vivo retention studies revealed higher corneal retention for co-crystals. In vivo studies on rat corneas demonstrated superior drug concentrations for riboflavin formulations compared to riboflavin-5-phosphate sodium, with hydrophilic gels showing prolonged corneal contact time. The THJ-TP formulation (Riboflavin-5-phosphate sodium and permeation enhancer (Transcutol P) containing hydrophilic gel formulations), containing riboflavin-5-phosphate sodium and Transcutol P, emerged as the most promising candidate. This research represents a significant advancement towards a non-invasive riboflavin-based treatment for keratoconus.