BACKGROUND: Chemotherapy preparation involves the use of specific techniques and equipment, given the need to maintain preparation sterility and its strict prescribed composition, and avoid occupational exposure to cytotoxic agents. OBJECTIVE: This study aims to identify the most relevant contents for pharmacy professionals' education and training programs and to elucidate the evaluation procedure these professionals should follow when handling cytotoxics. METHODS: We adopted the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines in conducting and reporting this systematic review. We conducted a literature search in PubMed, Cochrane, and LILACS to identify guidelines on cytotoxic drug preparation published between 2004 and 2024. Inclusion criteria included guidelines written in English, Spanish, or Portuguese that addressed the education, training, and/or evaluation of pharmacy professionals involved in handling cytotoxic drugs. We excluded guidelines developed for other health professionals (e.g. nurses) and guidelines exclusively addressing the manipulation of oral dosage forms. Citation searching was also performed to avoid search biases. Three researchers independently selected 20 guidelines that met the inclusion criteria, out of 3781 unique references identified. Four appraisers assessed the guidelines using the AGREE-II tool. RESULTS: Recommendations for training in cytotoxic drug handling generally included pre-initiation and periodic assessment. Personal protective equipment and engineering controls use, spill management, and aseptic technique were the most frequently mentioned specific training contents. We have developed a training proposal based on the guidelines, with four training levels that address the specific identified content. Each level presents potential competency assessment strategies. CONCLUSION: Included publications frequently recognized that conducting educational programs before and during the preparation of cytotoxic drugs was important and the combination of theoretical and practical learning seems of the utmost relevance. The development or update of guidelines in this area should prioritize their effective applicability to facilitate their implementation.