The present study aimed to prepare methotrexate-loaded transdermal patches with different blends of hydrophobic and hydrophilic polymers (Eudragit S-100 and hydroxypropyl methylcellulose) at different concentrations. The polymers employed in transdermal patches formulations served as controlled agent. Transdermal patches were prepared using the solvent casting technique. The suitable physicochemical properties were obtained from the formulation F5 (HPMC and Eudragit S-100 (5:1). Various penetration enhancers were employed in different concentrations to investigate their potential for enhancing the drug permeation profile from optimized formulations. A preformulation study was conducted to investigate drug–excipient compatibilities (ATR-FTIR) and the study showed greater compatibility between drug, polymers and excipients. The prepared patches containing different penetration enhancers at different concentrations were subjected for evaluating different physicochemical parameters and in vitro drug release studies. The obtained data were added to various kinetic models, then formulated patch formulations were investigated for ex vivo permeation studies, in vivo studies and skin drug retention studies. The prepared patches showed elastic, smooth and clear nature with good thickness, drug content, % moisture uptake and weight uniformity. The prepared transdermal patches showed % drug content ranging from 91.43 ± 2.90 to 98.37 ± 0.56, % swelling index from 36.98 ± 0.19 to 75.32 ± 1.21, folding endurance from 61 ± 3.14 to 78 ± 1.54 and tensile strength from 8.54 ± 0.18 to 12.87 ± 0.50. The formulation F5, containing a greater amount of hydrophilic polymers (HPMC), showed increased drug release and permeation and drug retention when compared to other formulated transdermal patch formulations (F1-F9). No significant change was observed during a stability study for a period of 60 days. The rabbit skin samples were subjected to ATR-FTIR studies, which revealed that polymers and penetration enhancers have affected skin proteins (ceramides and keratins). The pharmacokinetic profiling of optimized formulation (F5) as well as formulations with optimized concentrations of penetration enhancers revealed Cmax ranged 167.80 ng/mL to 178.07 ± 2.75 ng/mL, Tmax was 8 h to 10 h, and t1/2 was 15.9 ± 2.11 to 21.49 ± 1.16. From the in vivo studies, it was revealed that the formulation F5-OA-10% exhibited greater skin drug retention as compared to other formulations. These results depicted that prepared methotrexate transdermal patches containing different blends of hydrophobic and hydrophilic polymers along with different penetration enhancers could be safely used for the management of psoriasis. The formulated transdermal patches exhibited sustained release of drug with good permeations and retention profile. Hence, these formulated transdermal patches can effectively be used for the management of psoriasis.