The focus of current research work was to develop and validate size-exclusion chromatography method and develop and evaluate gel formulation of deferoxamine conjugated with PEGylated carbon nanoparticles (DEF-PEG-CNP) for topical delivery. Size-exclusion chromatography-based method was validated as per ICH guidelines. Effect of Carbopol® 974P and Transcutol® on the nanoparticles’ permeation was studied by 3-level full factorial design of experiment. Gel formulations were characterized for viscosity, cohesive and adhesive force by texture analyzer, and drug permeation through pig ear and human skin. The analytical method was specific as no interference from solvent or excipients were observed and met preset criteria of validation with limit of quantification of 0.24 ± 0.00 μg/mL. The nanoparticles permeation, steady state flux, and retained drug were statistically (p < 0.05) affected by Carbopol® 974P and Transcutol® percentage in the gel formulations. The permeation, steady state flux, and retained nanoparticles from the gel formulations varied from 23.2 ± 2.5 % to 70.9 ± 113.3 %, 0.8 ± 0.3 to 6.6 ± 2.1 μg/cm2.h, and 5.6 ± 0.3 to 38.8 ± 8.8 µg/g, respectively. Permeation of the nanoparticles was 1.9 folds higher in pig skin compared to human skin. Immunofluorescence detected successful permeation of DEF-PEG-CNP particles into skin. In conclusion, the analytical method can quantify the nanoparticles from the gel formulation without interference, and gel formulation of the nanoparticles can permeate across the skin.