Cell disaggregation is an essential step in preparing zebrafish embryos for flow cytometry, enabling the isolation of specific cell populations based on fluorescence markers. This protocol details the preparation of single-cell suspensions from zebrafish embryos, focusing on dissociation techniques to ensure that cells are viable and appropriately suspended for analysis. The method uses enzymes such as trypsin for cell dissociation and incorporates key steps like centrifugation, washing, and filtration to optimize sample quality for subsequent flow cytometry.