Anorectal cytology (ARC) is increasingly accepted as a valid screening tool for the diagnosis of squamous intraepithelial lesions in populations at increased risk for anal cancer. As with cervical cancer screening protocols, proper patient preparation, specimen collection and specimen processing are essential for obtaining an optimal cytological sample. With attention and experience, the clinician can collect the best possible ARC specimen for laboratory evaluation. The incorporation of repeated interval anal cytology into standard surveillance practices for high-risk individuals is a valuable tool for the early detection of human papillomavirus-related anal squamous epithelial lesions and the prevention of anal squamous cell carcinomas.