Abstract Human bladder cancer is one of the most expensive cancers to treat due to its high rate of reoccurrence. Development and characterization of animal models for human cancers is important for the improvement of diagnosis and therapy. The bladder transitional cell carcinoma (TCC) of domestic animals resembles human bladder TCC in many aspects, therefore cell lines derived from bladder TCC of dogs are valuable model for studying human bladder TCC. A chemotherapeutic agent, Doxorubicin (Dox), is often used to treat advanced bladder cancer. Despite the success of Dox based therapies, drug resistance and cardio-toxicity are limiting factors in treating any cancer with Dox. N-benzyladriamycin-14-valerate (AD198), a novel derivative of Dox has no measurable cardio-toxic effects in the rodent model of lymphoma. In this study, we focused in comparing the efficacy and mechanisms of Dox and AD198 in human and primary canine bladder transitional cell carcinoma (TCC) cells in vitro. Using human T24 and UMUC-3 TCC and three canine primary bladder K9TCC-Dakota, K9TCC-Lillie, and K9TCC-Molly cell lines, we evaluated the Dox and AD198 efficacy on cell proliferation by MTS assay. Caspase 3/7 assay, reactive oxygen species (ROS) production, and western blot analysis were used to study the mechanisms of Dox- and AD198-induced apoptosis. AD198 was more effective than Dox in inhibition of cell proliferation in tested TCC cells in dose-dependent manner. ROS production was increased when compared to control by both Dox and AD198 leading to apoptosis, which was confirmed by caspase 3/7 activity and cleavage of poly ADP ribose polymerase (PARP) in tested TCC cells. AD198 increased ROS production significantly more than Dox in all tested TCC cells. Both Dox and AD198 increased phosphorylation of pro-apoptotic p38 MAPK, while at the same time increased the activity of anti-apoptotic phosphatidylinositol 3-kinase (PI3K/AKT/mTOR) signal transduction pathway in tested TCC cells. When a PI3K inhibitor, LY294002, was used in combination with either Dox or AD198, cell proliferation was inhibited more effectively than when drugs used alone. In addition, cleaved PAPR was increased when TCC cells were co-treated with LY294002 and in combination with Dox or AD198 as compared to either drug alone. Our data suggest that AD198 as novel derivative of Dox may be a valuable treatment option for bladder TCC cancers. Dox- and AD198-induced AKT phosphorylation that is an indicator of pro-survival and drug-resistance mechanisms of chemotherapies in tested bladder TCC cancer. Combined therapy of Dox or AD198 with inhibitors of PI3K/AKT1 pathway might lead to more effective treatment outcome for human and veterinary patients diagnosed with bladder TCC cancer. Evaluation of new therapeutic and imaging drugs that have shown promise in vitro and in vivo in the rodent model are important; however, pet animals like, dogs and cats with naturally-occurring tumors provide an important step for successful translation from rodents to human clinical application. Citation Format: Dmitriy Smolensky, Kusum Rathore, Maria Cekanova, Maria Cekanova. Novel derivative of doxorubicin, AD198, for treatment of bladder transitional cell carcinomas in vitro. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C182.