Recently, several countries approved the use of cannabis flowering tops with standardized amount of ∆9-tetrahydrocannabinol (THC), cannabidiol (CBD) to treat several diseases. Therapeutic monitoring of medical cannabis products administered to patients for the established pathologies is rarely carried out. Previous few investigations have been developed in conventional matrices like blood and urine. This is the first study involving hair analysis of THC, CBD and their metabolites in patients treated with medical cannabis. An ultra-high-performance liquid chromatography-tandem mass spectrometry method to quantify THC, CBD, and metabolites, i.e., 11-nor-9-carboxy-THC (THC-COOH), 11-hydroxy-THC (11-OH-THC) cannabidiol-7-oic acid (7-COOH-CBD), 7-hydroxycannabidiol (7-OH-CBD), 6-α-hydroxycannabidiol (6-α-OH-CBD) and 6-β-hydroxycannabidiol (6-β-OH-CBD) in hair samples was developed and fully validated. The validation results indicated that the method was accurate (average inter/intra-day error, <10%), precise (inter/intra-day imprecision, <10%), and fast (10 min run time). Average hair concentrations in four patients treated with different formulations of medical cannabis were 2.75 ng/mg THC, 2.87 ng/mg 11-OH-THC, and 0.32 ng/mg THC-COOH (n = 3); 1.65 ng/mg CBD, 2.73 ng/mg 7-OH-CBD, 1.29 ng/mg 7-COOH-CBD, 0.35 ng/mg 6-α-OH-CBD, and 0.03 ng/mg 6-β-OH-CBD. The proposed method proved suitable for a fast and sensitive determination of all target compounds allowing high throughput testing in individuals monitored for medical cannabis treatments.