Background The use of injectable solutions for aesthetic purposes has increased tremendously, but lacks objective support. We aimed at assessing static and dynamic effects of botulinum toxin A (BoNTA) on glabellar lines by use of an objective three-dimensional methodology. Methods We prospectively collected three-dimensional stereographic photographs of two different facial expressions (pretreatment, 30 and 90 days posttreatment) in 21 patients, receiving a total of 20 units of BoNTA in both corrugator supercilii muscles. The primary endpoint was the three-dimensional static and dynamic surface irregularity, and secondary endpoints were the glabellar line scale and overall patient satisfaction. Blinded retrospective data analysis and statistical evaluation were performed with p  < 0.05 considered statistically significant. Results Static glabellar lines (neutral facial expression) were significantly reduced by − 17% and − 24% on day 30 and 90 posttreatment, respectively (vs. pretreatment; both p  < 0.0001). Dynamic glabellar frown lines (firmest possible bilateral eye closure) demonstrated a reduction of surface irregularity by − 26% and − 21% on day 30 and 90 posttreatment, respectively (vs. pretreatment; both p  < 0.0001). The subjective dynamic glabellar line scale documented a statistically significant improvement on day 30 posttreatment (mean ± SD: 1.5 ± 0.8; p  < 0.05) versus pretreatment (2.8 ± 1.0). Polled patients confirmed a subjective wrinkle improvement 90 days posttreatment. Conclusion The presented setup detected even subtle changes of BoNTA treatment for facial wrinkling and is a promising asset for scientific evaluations of clinical studies analyzing the outcome and duration of efficacy of injectable solutions on the face. Level of Evidence IV This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .