The main item routes of antibiotics in the environment are the application of organic wastes to improve soil quality and the irrigation with recycled water. Once in the environment, antibiotics can be introduced in the food chain through their uptake by crops. This paper describes the development of an analytical method based on ultrasound-assisted extraction for the determination of seven antibiotics in lettuce. The developed method was applied to evaluate antibiotic uptake by lettuce grown in pots fertilized with composted poultry litter doped with a mixture of antibiotics to reach a final concentration of 2.5 µg/g in soil. Lettuce were harvested after 21, 36, and 55 days. Five of the seven studied antibiotics were found in all samples. The highest uptake was found for lincomycin (51 ng/g fresh weight) followed by sulfamethoxazole (44 ng/g fresh weight) and sulfamethazine (21 ng/g fresh weight) in lettuce harvested after 21 days. An important decrease of their levels was observed after 36 days, but these levels remained similar after 55 days. Although levels found in lettuce were low, the presence of antibiotics demonstrates the need for further assessing food safety risks related with the use of soil amendments or irrigation water contaminated with antibiotics.