Background Vitiligo is a depigmenting disorder of the skin and mucous membranes. Latanoprost (LT) is a prostaglandin F2α analog used in the treatment of glaucoma and was found to induce skin pigmentation. Aim This comparative study aims to assess efficiency of topical 0.005% LT solution in combination with microneedling for treatment of segmental vitiligo. Patients and methods The authors addressed 50 patients with segmental vitiligo stable for at least one year divided into two groups in this study: group A included 25 patients treated with topical 0.005% LT solution in combination with microneedling sessions by using dermapen, and group B included 25 patients who were treated with topical 0.005% LT solution alone without microneedling. The response to treatment was evaluated by serial photographs, degree of pigmentation, and vitiligo area scoring index (VASI). All patients were evaluated before the start of the treatment and each month after the end of the treatment. Results The group A, which was treated by combination of topical 0.005% LT ophthalmic solution and microneedling, showed significant improvement in VASI score, whereas group B, which was treated by topical 0.005% LT ophthalmic solution alone without microneedling, showed minimal improvement in VASI score. Conclusion The use of microneedling in combination with using topical LT improves the results of treatment of segmental vitiligo.