Background: Currently, cancer is among the leading causes of morbidity and mortality in the world. Exposure to CDs may occur during drug preparation and mixing, during drug administration, during transport, and cleaning spills and waste disposal. Healthcare workers who prepare or administer antineoplastic drugs, or who work in areas where these drugs are used, can be exposed to these agents. This also affects the public around the exposed area if appropriate disposal system is not known. Several studies reported increased risks of leukemia and breast cancer among nurses handling CDs and not following safety guidelines. Because of the absence of studies in Ethiopia, the current study was conducted to determine the knowledge level of cytotoxic drug handling and associated factors among health professionals in the University of Gondar Comprehensive Specialized Hospital. Methods: The institutional-based cross-sectional study was conducted from June to August 2019. Epi info 7.1 was used for data entry and then exported into SPSS version 20 for computing, recording, and statistical analysis. Logistic regression was used to explain the relationship with independent variables. Results: Four hundred and twelve health professionals participated in the study with 53.4% males. The participants' mean age was 29.9 (± 5.43) years ranging from 20-60. Two hundred and twenty-three (54.1%) health professionals heard about cytotoxic drugs, and 52.7% (95% UI 47.8-57.8%) had good knowledge of cytotoxic drug handling. Being male sex (AOR = 1.84, 95% CI (1.13-3.00)), age of 29-31 (AOR = 1.99, 95% CI (1.03-3.84)), hearing information about cytotoxic drug handling (AOR = 2.53, 95% CI (1.43-4.47)), ever attended training on cytotoxic drug handling (AOR = 3.15, 95% CI (1.13-8.79)), ever taking courses related to cytotoxic drugs (AOR = 2.03, 95% CI (1.15-3.59)), and good practice (AOR = 3.24, 95% CI (1.95-5.37)) were significantly associated with knowledge towards cytotoxic drug handling. It is therefore imperative to train health professionals and to incorporate CDs handling related course contents while revising curricula to raise the knowledge of health professionals about proper cytotoxic drug handling.(Continued on next page) Conclusion: Above half of the study participants scored higher than the median of the cytotoxic drug handling knowledge questions. Sex, age, information about cytotoxic drug handling, training, taking courses related to cytotoxic drugs, and good practice were significantly associated with knowledge towards cytotoxic drugs handling.