A sensitive method has been developed for determination of trace leaching phthalate esters (PAEs) in water and urine from plastic containers by direct immersion solid-phase microextraction (DI-SPME) coupled to gas chromatography-mass spectrometry (GC-MS). The limits of detection (LODs) for PAEs in water and urine had values between 0.2–8 ng L−1 and 4–70 ng L−1, respectively. The proposed method was successfully used for quantification of trace PAEs in water in microwavable polypropylene containers heated by microwave irradiation and in the urine of patients treated with a glucose injection using polyvinyl chloride tubing.