Mycotoxins, toxic metabolites of fungi (e.g., Aspergillus, Fusarium) in optimal temperature/humidity, contaminate a large range of global crops and animal feed. They cause multi-toxicity (e.g., carcinogenicity), harm animal health, enter the human food chain via animal products (e.g., milk, meat), and damage the agricultural economy, making accurate detection critical. This chapter reviews mycotoxin detection in animal feed, covering sample collection/preservation/pretreatment, detection methods (chromatographic, rapid, emerging technologies), regional contamination, and masked mycotoxin detection. Future directions focus on multiplex detection, eco-friendly methods, and data standardization to safeguard feed safety and the food chain.