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Metabarcoding-based diet analysis is a valuable tool for understanding the feeding behavior of a wide range of species. However, many studies using these methods for wild animals assume accuracy and precision without experimental evaluation with known positive control food items. Here, we conducted a feeding trial experiment with a positive control community in pasture-raised chickens and assessed the efficacy of several commonly used DNA extraction kits and primer sets. We hand-fed 22 known food items, including insects and plants, to six backyard laying hens and collected their excreta for eight h. We evaluated the efficacy of three DNA extraction kits, three primer sets for plant identification (targeting rbcL, trnL, and internal transcribed spacer 2 [ITS2]), and three primer sets for arthropod identification (targeting cytochrome oxidase subunit I [COI]). The detection success rate of our positive control food items was highly variable, ranging from 2.04% to 93.88% for all kit/primer combinations and averaging 37.35% and 43.57% for the most effective kit/primer combination for plants and insects, respectively. Extraction kits using bead-based homogenization positively affected the recovery proportion of plant and insect DNA in excreta samples. The minimum time to detect known food items was 44 min post-feeding. Two COI primer sets significantly outperformed the third, and both recovery proportion and taxonomic resolution from ITS2 were significantly higher than those from rbcL and trnL. Taken together, these results display the potential variability that can be inherently present in DNA-based diet analyses and highlight the utility of experimental feeding trials in validating such approaches, particularly for omnivores with diverse diets., (© 2024 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)