This study aimed at develop and validate the procedure for collecting exhaled breath for drug testing. Patients receiving methadone maintenance treatment were recruited for the study. Methadone levels were measured using liquid chromatography- electrospray-tandem mass spectrometry. The sampling device was based on a 47-mm C(18) filter and used under pressure to aid flow through the filter. The mouth was rinsed before sampling, and the device was constructed to protect against any saliva contamination. Methadone was present in breath samples before and after the daily intake of methadone. The mean (± SD) pre-dose level was found to be 135 ± 109 pg/min (n = 48, median 121). The exhaled methadone increased after dose intake. Saliva levels of methadone were high in comparison with exhaled breath levels. Saliva contamination was suspected in about 10% of the collected samples. Similar results were obtained using 1, 3, and 10 min sampling times. The inter- and intraindividual variability were found to be similar and in the order of 50%. Alternative sampling using XAD-2 beads and solid-phase microextraction fiber was found to be possible and enables sampling with low back pressure and with no need for pump assistance. The presented results confirm that breath testing is a new possibility for the detection of drugs of abuse.