Ensuring asepsis of the surgical site before surgery is an essential component of safe surgical practices to reduce the incidence of surgical site infections in veterinary medicine. The current accepted method of skin preparation is a multistep process that alternates either a povidone-iodine or chlorhexidine soap scrub with a 70% alcohol rinse. After cleansing, the site is left to dry before draping. The goal of this study was to assess the effectiveness of a waterless alcohol-based (WAB) antiseptic as part of a 2-step procedure after the soap scrub. WAB antiseptics are commonly used as a presurgical hand scrub for the surgeon as they evaporate quickly and provide effective antisepsis. Previous studies have examined the WAB antiseptics in small animal surgeries. We tested this approach in large animal surgery. Twenty-four rhesus macaques were divided into 4 groups that received one of the following treatments: saline and alcohol, iodine-alcohol-iodine, soap scrub/WAB, and chlorhexidine-alcohol-chlorhexidine. The surgical site was swabbed before and after treatment and plated to assess sterility. Overall, no colonies were recovered from skin treated with WAB antiseptic, establishing it as an effective alternative to the current standard protocol. This method will simplify the current 3-step procedure and reduce animal handling, the use of materials, and the time necessary for surgical preparation.