The purpose of this study was to determine the permeation of the hydrophilic compound 5-fluorouracil through human epidermal membranes, Ehrlich Ascites Carcinoma (EAC) cells were used as a model cell line to evaluate the cytotoxic concentration and anti-tumor activity of 5-fluorouracil (5-FU) through transdermal drug delivery for tumors. Cytotoxicity was assessed by exposing cell suspension to increased concentration of drug from 20-100 μg/ml and measuring the viable cell count by tryphan blue exclusion method. Results confirmed that 100 μg/ml of 5-FU was cytotoxic. The increase in the life span (ILS) was 87.05% with maximum survival time of 30.5±1.87 days. For 5-fluorouracil monolithic matrix transdermal patch, the results were statistically significant (p < 0.05) compared to untreated control, anti-tumor activity was very effective compared to intravenous therapy. Patches did not show any sign of erythema, vesiculations or bullaous reaction. Mean cumulative skin irritation and adherence scoring for both animal and humans proved that none of the irritation sensitization reactions score were zero and less than one, while good adherence score was 0, with complete adherence to the skin, without leaving any adhesive residue on skin with scores = 0 in human subjects. Transdermal patches showed 100% flatness, thickness 150±0.03 mm, good content uniformity, folding endurance ( > 500 foldings), smoothness, transparency, flexibility and appearance. Pharmacokinetic studies of 5-FU transdermal patch in rabbits showed half-life 95±0.5 min, Cmax (ng/ml) 863.25,AUC0-α (ng/ml/h)1567±36 and Tmax(h) 1.5 with controlled release for 24 h which was very significant (p < 0.001) compared to intravenous route. Recent patents has been reported for suitable treatment of tumors and cancer, by topical and transdermal applications. Velcro protection jackets were suitable for this study and protected our applied transdermal patched from being licked, scratched and rubbed off.