Abstract Cellular senescence is an emerging hallmark of cancer. Senescent cells can lead to tumor development and chronic inflammation that stimulate age-related diseases. The secretion of senescent cells also generates an inflammatory environment that promotes tumor growth. Senolytics are pharmaceutical agents that selectively eliminate senescent cells through various mechanisms. Identifying the mechanism is crucial for the further development of senolytic drugs. ABT263 (also named navitoclax), a Bcl-2 and Bcl-xl dual inhibitor, is a well-known senolytic agent. Recently the first Bcl-2 and Bcl-xl dual-targeting proteolysis-targeting chimera (PROTAC), 753b, was developed, which has increased potency against senescent cells but minimized platelet toxicity. EF24 is another senolytic agent but the direct target is still unclear. Comprehensive analysis and comparison of the media environments from young cells, senescent cells, and senescent cells dosed with different senolytics can contribute toward solving senolytic mechanisms and make the drug discovery process more efficient and effective. WI38 cell culture media was used to grow young, senescence, and senolytic (753b or EF24)-dosed senescent cells for 72 hours. Cell culture media was collected at 10 different growth time points, subjected to protein crash using acetonitrile, and diluted with water prior to analysis. A 17-minute liquid chromatography triple quadrupole mass spectrometry method was developed for senescent cell and senolytic-added senescent cell culture media analysis. One hundred and forty-five cell culture related compounds including amino acids, nucleic acids, metabolites, sugars, and vitamins were simultaneously analyzed. A relative concentration trend graph was generated as the result for each of the cell growth related analytes. Thirteen compounds exhibited trends that distinguished at least one experimental group from the others in the 72 hours growth period. These compounds were asparagine, 5’-Methylthioadenosin, saccharopine, serotonin, alanyl-glutamine, urocanic acid, o-phosphoethanolamine, hypoxanthine, xanthosine, uracil, pyridoxal, nicotinic acid, and pyruvic acid. The differences observed in the cell environment reflect the differences of the activated pathways and mechanisms between the young, senescent, and senolytics-dosed senescent cells. This rapid and multicomponent cell culture analysis method enables scientists to analyze the environment as senescent cell and senolytic-dosed senescent cells grow. Comparing the chemical profile in the cell environments can be a potential strategy in understanding the mechanisms of the senolytic drugs. This information may also be valuable in predicting the toxicity and side effects of the new senolytic drugs. Citation Format: Evelyn Wang, Stephen Kurzyniec, Ethan Hain, Yoshiyuki Okamura, Yang Yang, Dongwen Lv. Characterizing cellular senescence response to senolytic compounds using cell culture media analysis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4792.