Objective - To establish 2 vaccine-associated feline sarcoma (VAFS) cell lines and to determine their in vitro sensitivity to the chemotherapeutic agents doxorubicin and mitoxantrone. Sample Population - Tumor specimens collected from 2 cats undergoing surgery for removal of vaccine-associated sarcomas. Procedures - Tumor specimens were minced and treated with trypsin under aseptic conditions to obtain single-cell suspensions, which were then cultured in vitro in medium supplemented with 5% heat-inactivated fetal bovine serum. Growth rates and sensitivity after 24 hours of exposure to various concentrations (0.1 to 100 μg/ml) of doxorubicin and mitoxantrone were assessed for each cell line. Survival of cells was estimated 3 days after exposure to the 2 agents, and the concentration of each drug that resulted in a 50% reduction in the number of viable cells (IC50) was calculated. Results - Two tumor-derived cell lines (FSA and FSB) were successfully established and determined to be sensitive to doxorubicin and mitoxantrone. Under the conditions tested, the IC50 of doxorubicin were 0.6 and 1.5 μg/ml for cell lines FSB and FSA, respectively. The IC50 of mitoxantrone was 0.4 μg/ml for both cell lines. Conclusions and Clinical Relevance - The establishment of VAFS cell lines provides a tool for the in vitro screening of antitumor drugs. Doxorubicin and mitoxantrone were effective in decreasing the number of viable cells in the 2 cell lines tested. Both of these anthracycline antibiotics have been used to treat various neoplasias in cats, and their efficacy for adjuvant treatment of vaccine-associated sarcomas should be further evaluated.