IMPORTANCE Smiling can be a voluntary or involuntary movement. Facial reanimation procedures differ in their ability to restore a spontaneous smile, and an assay designed to evoke and evaluate a spontaneous smile is not available. OBJECTIVE To develop and validate an assay to assess the spontaneous smile of patients with facial paralysis. DESIGN, SETTING, AND PARTICIPANTS This was an exploratory cohort study. A series of short video clips were administered to laypersons via an online survey service from January 1, 2014, to March 31, 2014. Respondents rated how funny each video was on a visual analog scale from 0 to 100. The 4 funniest videos were selected to generate a 1½-minute spontaneous smile assay. The assay was then administered from July 1, 2014, to December 31, 2014, to 2 different study groups: the first was composed of 100 healthy individuals (control group) and the second was composed of 30 patients with facial paralysis. We analyzed the capability of this assay to provoke at least 1 spontaneous smile and calculated smile excursion in both groups. Statistical analysis was performed using analysis of variance. INTERVENTION Spontaneous smile assay administered to both healthy and diseased groups. MAIN OUTCOMES AND MEASURES Ability of the assay to elicit smiles, as defined by an oral commissure excursion greater than 3 mm, as well as difference in commissure excursion. RESULTS Ninety-five (95.0%) participants in the control group and 29 (96.7%) patients with facial paralysis experienced at least 1 oral commissure excursion that appeared to be a spontaneous smile while viewing the assay. Mean oral commissure excursion with spontaneous smile was 9.08 mm (95% CI, 2.77-15.39) in controls, 6.72 mm (95% CI, 3.13-10.31) on the healthy side in patients with flaccid facial paralysis (P=.004 vs controls), and 9.64 mm (95% CI, 3.52-15.76) on the healthy side in patients with nonflaccid facial paralysis (P=.74). Among patients with flaccid facial paralysis, a statistically significant difference was found between smile excursion of the affected and the unaffected sides (P = .03). There was no statistically significant difference in the measurement between sides for the control group (P = .67). CONCLUSIONS AND RELEVANCE Although humor is a challenging construct to universalize, our assay was able to elicit a smile in almost all individuals in the group with facial paralysis and the control group. The spontaneous smile assay will facilitate future research on the ability of facial reanimation procedures and other interventions to restore a spontaneous smile. LEVEL OF EVIDENCE NA.