People diagnosed with substance use disorder (SUD) might represent a high-risk subpopulation for New Psychoactive Drugs (NPS) consumption, and hair analysis offers a unique perspective to assess drug prevalence in this population. The present study aims to assess the prevalence of NPS and their co-consumption with traditional drugs of abuse (DoA) in individuals diagnosed with SUD. Hair samples from patients under care at the addiction treatment service of Bologna, Italy, for a diagnosed SUD, were collected during 2023 and analyzed by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS), using a previously validated method. Among the 88 patients included, 95.5% tested positive for at least one substance, of which 88.1% for traditional DoA only, and 11.9% for NPS in addition to DoA. Among the positive samples, patients were found positive for more than two drugs in 67.9% of cases. The combination of DoA and NPS was more frequent in the younger age group (<
21-30 years old, compared to 31-70, p = 0.025). Ketamine was detected in 8.0% of all samples, with mean hair levels 49.68 pg/mg (ranging 8.55-81.90 pg/mg) and was frequently accompanied by cocaine (85.7% of cases). Fentanyl was detected in 3.4% of all samples, while, among NPS, buphedrone was the only one detected. Our retrospective study highlights that the consumption of NPS is relatively low compared to other vulnerable or high-risk populations. However, the prevalence of polydrug consumption and the high rate of ketamine-cocaine combination warrant careful monitoring even in this population.