Usnic acid is a unique lichen metabolite of industrial importance, widely studied to explore its pharmacological potential and valued especially as an antibacterial agent in cosmetics. Although a vast number of papers describe usnic acid extraction from various lichen species, none has so far provided an unequivocal indication of the best extraction procedure for this compound. Thus, the current study was focused on the direct comparison of three commonly used usnic acid extraction methods (heat reflux, shaking, ultrasound-assisted extractions), which were optimized using fractional factorial design. Heat reflux extraction, shaking extraction and ultrasound-assisted extraction were first optimized in a series of experiments using fractional factorial design, with respect to three parameters: the extraction time, the solvent used and the number of extraction repetitions. HPLC was employed for usnic acid quantitative analysis. The best scores for each extraction method were statistically compared and the optimal conditions were indicated. The optimal set of parameters for usnic acid was established to be a single, 60 min heat reflux extraction with acetone. This extraction scheme provided 4.25 ±0.08 mg g−1 d.w. of usnic acid, while for ultrasound-assisted and shaking extractions the amount was two- or even four times lower (2.33 ±0.17 and 0.97 ±0.08 mg g−1 d.w., respectively). The optimal procedure for usnic acid extraction described here may be suitable for effective acquisition of this compound for scientific research purposes, but also for applications in the pharmaceutical or cosmetic industries.