This work describes two methods for rapid and simple preparation of urine samples for determining the 239Pu content. The preparation of the samples was carried out by evaporation or coprecipitation of Pu, followed by acidic digestion in a microwave oven. The concentration and purification of Pu were achieved by using ion exchange resins AG1X2 and AG1X8 (Bio-Rad Laboratories). The quantification of 239Pu was carried out employing three different instrumental techniques, the more conventional Alpha Spectrometry (AS), and two mass spectrometry methods: High-Resolution Inductively Coupled Plasma Mass Spectrometry (HR-ICP-MS) and a low-energy Accelerator Mass Spectrometry (AMS). The chemical yield was determined by AS being, on average, 76 ± 7%. The minimum detectable activity (MDA) for AS, HR-ICP-MS and AMS was, respectively, about 50 fg (∼0.1 mBq), 23 fg (∼0.05 mBq) and 0.44 fg (∼1 µBq) per sample, respectively. The results presented in this work demonstrate the excellent performance and suitability of the mass spectrometric techniques for the routine measurement of 239Pu at ultratrace levels in human urine samples, and also for the reassessing of AS planchets if it were needed. The application of HR-ICP-MS and low-energy AMS reduces the sample processing time, and helps us to improve the laboratory productivity, also giving better MDA values when compared to the classical radiometric methods.