Background Substance use is a global health concern, with opioids contributing significantly to the disease burden. In Kenya, Medically Assisted Therapy (MAT) programs using methadone have been implemented to address opioid use disorder. Despite the effectiveness of methadone, the concurrent use of other substances remains a critical challenge. This study aimed to assess substance use patterns at enrolment and evaluate the prevalence of negative urine opioid screens among patients attending a MAT clinic at a tertiary hospital in Kenya. Methods This retrospective cohort study analyzed data from the medical records of 713 patients enrolled in the MAT clinic between December 2014 and February 2018. Data on sociodemographic characteristics, concurrent substance use at enrolment, and urine opioid screen results at 6, 12, 18, and 24 months were collected. Multivariate analyses were performed to identify factors associated with concurrent substance use, and the likelihood of achieving negative urine opioid screens. Results At enrolment into the MAT program, nearly all participants (712 out of 713) reported concurrent use of additional substances, with tobacco (91%) and cannabis (82.9%) being the most common. Concurrent substance use was significantly influenced by participants age, gender, education level, and route of administration of the substance. The program achieved an 81.3% retention rate at 24 months. However, the prevalence of negative urine opioid screens was lower compared to other populations, with a gradual increase from 61.3% at 6 months to 81.4% at 24 months. Notably, male patients (HR = 1.411, 95% C.I. 1.063–1.873, p = 0.01700) and those receiving higher methadone doses (HR 7.052, 95% CI 3.408–14.593, p<0.0001) were more likely to achieve negative urine opioid screens. Conclusions This study reveals a high prevalence of concurrent substance use among patients enrolling in the MAT program in Kenya, which may affect their likelihood of achieving negative urine opioid screens. These findings underscore the need for methadone treatment programs to adopt comprehensive approaches that address all substance use disorders to improve treatment outcomes.