Analyzing urine is common in drug-facilitated sexual assault cases if the analysis of blood is not optimal. The efficient enzymatic pretreatment of urine is important for cleaving glucuronides and improving the detection of the parent drug. The aim was to investigate the efficiency of three β-glucuronidases on eleven glucuronides relevant to DFSA at different incubation periods and temperatures. Human drug-free urine was fortified with 11 glucuronides, hydrolyzed with either β-glucuronidase/arylsulfatase (Helix Pomatia), recombinant β-glucuronidase B-One™ or recombinant β-glucuronidase BGTurbo™ and incubated for 5, 10, 60 min, 18 h and 24 h at 20 °C/40 °C/55 °C before UHPLC–MS/MS analysis. The stability of 141 drugs and metabolites relevant to DFSA was investigated by incubating fortified urine under the same hydrolysis conditions. B-One™ showed efficient hydrolysis (>90%) of most glucuronides in 5 min at all temperatures, while BGTurbo™ showed a similar efficiency (>90%), but the optimal temperature (20–55 °C) and incubation time (5–60 min) varied among analytes. The β-glucuronidase/arylsulfatase had the lowest efficiency and required the longest incubation (24 h) at 40–55 °C. The stability of 99% of 141 drugs and metabolites was not affected by incubation at 20–55 °C for 24 h. Recombinant enzymes show promising results for the simple and efficient hydrolysis of a broad panel of glucuronides relevant for DFSA.