This protocol was developed for the human breast cell atlas (HBCA) project to obtain high-viability cell suspensions from freshly dissociated breast tissues from human patients. There are two options for performing this protocol: rapid-dissociation (15-30 min) or exhaustive dissociation (overnight). In our experience the rapid dissociation protocol will provide higher cell viability and better representation of breast cell types, however the total number of cells dissociated may be lower after enumeration. In contrast the exhaustive protocol provides higher cell numbers and maintains good cell viability but may result in skewing of cell type representations over the long time period. The rapid-dissociation protocol was used to generate most of the cell suspensions for the MD Anderson human breast cell atlas project and has been optimized and tested more extensively.