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Poly (vinyl alcohol) microneedles were fabricated, characterized, and applied to enhance in vitro transdermal delivery of doxorubicin. The microneedles were fabricated using the micromolding technique with the drug load in different locations within the needle array. The polymer solution was assessed for rheological properties, drug dissolution, and chemical structurestudies. Microneedles (unloaded) and drug-loaded microneedles were characterized by optical microscopy, fluorescent microscopy, scanning electron microscopy, and drug release kinetics. Successful microporation of dermatomed human cadaver skin was demonstrated by dye binding, pore uniformity, histology, confocal laser microscopy, and skin integrity studies. The microneedles-mediated transdermal delivery of doxorubicin was investigated using vertical Franz diffusion cells.The fabricated microneedles were sharp, strong, and uniform. In vitro permeation studies showed that the microneedle-treated skin (4351.55 ± 560.87 ng/sq.cm) provided a significantly greater drug permeability than the untreated group (0.00 ± 0.00 ng/sq.cm, n = 4, p < 0.01). The drug location within the needle array was found to affect the drug release profile as well as its permeation into and across human skin.Skin microporation achieved by poly (vinyl alcohol) microneedles was found to enhance transdermal delivery of doxorubicin in vitro.