To determine the incidence of toluidine blue positive findings after speculum examination of sexual assault victims, we performed a prospective before and after study of 27 female patients presenting after sexual assault to a free-standing nurse examiner clinic. Before the insertion of a speculum, a 1% aqueous solution of toluidine blue was applied to the posterior fourchette and photographs were taken using colposcopy with digital imaging (16 × magnification). After the forensic examination was completed, dye was reapplied. Photographs taken before and after speculum examination were reviewed by three EM physicians for superficial lacerations or abrasions. Before speculum examination, genital injuries from sexual assault were documented in 67% of the patients (mean number of genital injuries, 1.4). After speculum examination, one patient (3.7%) demonstrated a new genital injury—an abrasion to the labia. Until further studies are performed, use of a speculum should be delayed until after toluidine dye application.