INTRODUCTION: Most forms of congenital and acquired deafness result from damage to cochlear hair cells or their associated neurons. In non-adherent culture conditions, neonatal tissue from the organ of Corti harbors cells that are able to generate floating clonal colonies, called otospheres. Otospheres demonstrate the capacity for self-renewal, proliferate, and express stem/progenitor cell (SPC) markers. The standardization of cell culturing procedures has become an essential tool in any laboratory working with SPC. OBJECTIVE: To standardize the proliferation assay for suspension culture of SPC from the organ of Corti of neonatal mice. We intended to compare 2 different proliferation assay test kits to determine the most appropriate method. METHODS: Cultures were maintained for 5 days in vitro in medium; we tested both kits (BrdU Labeling Kit Detect, Roche; and Click-iT® EdU cell proliferation assay, Invitrogen) at different concentrations and using diverse exposition times for the cell proliferation measurement components. RESULTS: We observed a toxic effect of BrdU and EdU components on the suspension culture. The best results in terms of viability and proliferation were obtained with a 0.1 mM/mL concentration of the final components in the culture medium, and with a maximum time of exposition of 24 h. CONCLUSION: The results of each test performed proved the effectiveness of both sets of proliferation assays used, based on staining signal capture by indirect immunofluorescence. Nevertheless, we noticed greater convenience using the kit from Roche because of the ease of preparation of the working solutions and the shorter time required for the experiment conclusion.