Ablative fractional lasers enhance uptake of topical therapeutics and the concept of fractional laser-assisted drug delivery has now been taken into clinical practice. Objectives We systematically reviewed preclinical data and clinical evidence for fractional lasers to enhance drug uptake and improve clinical efficacy. Methods We searched PubMed and Embase databases; 34 articles met the inclusion criteria. Studies were categorized into experimental preclinical studies and clinical trials, the latter graded according to level of evidence. Results All preclinical trials (n = 16) documented enhanced topical drug uptake into skin after ablative fractional laser treatment. Clinical evidence encompassed 18 studies, of which 9 were randomized controlled trials and 2 were controlled trials, examining neoplastic lesions, photodamaged skin, scars, onychomycosis, and topical anesthetics. The highest level of evidence was reached for actinic keratoses treated with methylaminolevulinate for photodynamic therapy (level IB, 5 randomized controlled trials), substantiating superior and long-lasting efficacy versus conventional photodynamic therapy. No adverse events were reported, but ablative fractional laser-assisted drug delivery implies risks of systemic drug absorption, especially when performed over large skin areas. Conclusions Fractional laser-assisted drug delivery is beneficial in enhancing preclinical and clinical outcomes for certain skin conditions.