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The ability to acquire chemical defenses through the diet has evolved across several major taxa. Chemically defended organisms may need to balance chemical defense acquisition and nutritional quality of prey items. However, these dietary preferences and potential trade-offs are rarely considered in the framework of diet-derived defenses. Poison frogs (Family Dendrobatidae) acquire defensive alkaloids from their arthropod diet of ants and mites, although their dietary preferences have never been investigated. We conducted prey preference assays with the Dyeing Poison frog ( Dendrobates tinctorius ) to test the hypothesis that alkaloid load and prey traits influence frog dietary preferences. We tested size preferences (big versus small) within each of four prey groups (ants, beetles, flies, and fly larvae) and found that frogs preferred interacting with smaller prey items of the fly and beetle groups. Frog taxonomic prey preferences were also tested as we experimentally increased their chemical defense load by feeding frogs decahydroquinoline, an alkaloid compound similar to those naturally found in their diet. Contrary to our expectations, overall preferences did not change during alkaloid consumption, as frogs across groups preferred fly larvae over other prey. Finally, we assessed the protein and lipid content of prey items and found that small ants have the highest lipid content while large fly larvae have the highest protein content. Our results suggest that consideration of toxicity and prey nutritional value are important factors in understanding the evolution of acquired chemical defenses and niche partitioning.