The International Society of Analytical Cytology (ISAC) Biohazard Working Group presents guidelines for sorting of unfixed cells, including known biohazardous samples, using jet-in-air, deflected-droplet cell sorters. There is a risk that personnel operating these instruments could become exposed to droplets and aerosols containing biological agents present in the samples. The following guidelines can aid in the prevention of exposures of laboratory personnel to pathogens contained in the sort samples. The document provides biosafety recommendations for sample handling, operator training and protection, laboratory facility design, and instrument setup and maintenance. In addition, it describes in detail methods for assessment of instrument aerosol containment. Recommendations provided here may also help laboratories to obtain institutional and/or regulatory agency approval for sorting of unfixed and known biohazardous samples.