In the current study, buprenorphine (BUP) and its major metabolite, nor-buprenorphine (NBUP), were determined in hair samples from former heroin users following Suboxone® treatment. Hair samples from 36 subjects were analyzed. The drugs of interest were isolated from hair by solid-liquid extraction with methanol and were determined by liquid chromatography-mass spectrometry, using an electrospray ionization interface. The analytical parameters of the method (such as linearity, limits of quantification, recovery, accuracy, and precision) were determined. The inter-quartile range of BUP levels was from 11.4 to 37.4 pg/mg (mean value 56.6 pg/mg) for the proximal hair segment, from 5.8 to 43.3 pg/mg for the middle hair segment (mean value 25.3 pg/mg), while a range from 4.3 to 33.9 pg/mg (mean value 105.2 pg/mg) for the distant to the root hair segment was determined. For NBUP the corresponding inter-quartile range was from 27.0 to 147.6 for the proximal segment (mean value 95.4 pg/mg), from 21.5 to 164.7 pg/mg for the middle segment (mean value 102.0 pg/mg) and from 20.4 to 103.6 pg/mg for the distant segment (mean value 156.8 pg/mg). The mean BUP/NBUP concentration ratio was 0.5. The daily dose of Suboxone® correlated significantly with BUP and NBUP levels in hair (p = 0.001 and p = 0.023) as well as with the BUP/NBUP ratio (p = 0.010). No significant correlation was found between the levels of BUP and NBUP and the duration of Suboxone® administration. The developed and validated method was successfully used for the determination of BUP and NBUP in hair samples collected from former heroin users under Suboxone® treatment.