High concentrations of aerosols can be generated within the sort collection area of cell sorters during instrument failures that cause the stream to deviate, such as a partial nozzle obstruction. Complete containment of these aerosol particles becomes essential for operator safety when working with potentially infectious or hazardous samples. Currently, aerosol containment is accomplished through the generation of continuous negative airflow within the sort collection area using an aerosol evacuation system, which can be enhanced by using primary containment devices such as biosafety cabinets. Unlike biosafety cabinets, many aerosol evacuation systems are not certified or tested on a regular basis after installation. Therefore, proper function of the system must be verified by the user prior to running hazardous samples to ensure that it is operational and provides sufficient protection for the operator. This protocol describes an updated procedure for verifying the containment and evacuation of aerosols generated when the stream is disrupted during an instrument failure. In this procedure, aerosols are generated to simulate a partial nozzle obstruction while running 1-µm fluorescent beads. Air samples are collected just outside the sort collection area using a disposable impactor-style aerosol sampler cassette and are examined for the presence of beads in an effort to detect aerosols. If no beads are present, aerosols were adequately contained and evacuated by the aerosol evacuation system. The presence of beads, however, indicates a potential failure of the aerosol evacuation system and/or other engineering controls that could result in the exposure of laboratory workers to any infectious or hazardous samples that are run through the instrument. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: Validation of the aerosol evacuation system using 1-µm fluorescent beads and disposable aerosol sampler cassettes Support Protocol: Preparation of 1-µm fluorescent bead reference slide.