There are few systematic studies evaluating injectable sedation protocols in reptiles, especially in aquatic turtle species. The goal of this study was to examine the efficacy of a commonly recommended injectable drug combination in reptiles, dexmedetomidine-midazolam, in red-eared sliders. An additional goal was to identify differences in sedation efficacy based on the location of injection. We hypothesized that forelimb injection of dexmedetomidine-midazolam would cause a greater depth of sedation compared to hindlimb injection. Eight healthy adult red-eared sliders were administered forelimb and hindlimb, subcutaneous (SC) injections of dexmedetomidine (0.1 mg/kg) and midazolam (1.0 mg/kg) in a blinded, randomized, crossover study. To gauge sedation level, we compared various reflexes at set intervals, including muscle tone, jaw tone, cloacal tone, and pelvic limb withdrawal reflex. Heart rate and respiratory rate were monitored throughout sedation and the ability to perform venipuncture was simulated in each sedated turtle at 30 minutes post-injection. Turtles were administered atipamezole and flumazenil SC for reversal at 45-minutes post sedation injection. Dexmedetomidine-midazolam produced moderate sedation characterized by reduced muscle, jaw and cloacal tone. Neck extension and needle prick for simulated venipuncture possible in 8/8 turtles in the forelimb treatment and 7/8 turtles in the hindlimb treatment. Mean time to recovery was 70 ± 32 min and 85 ± 54 min for forelimb and hindlimb treatments, respectively. Injection site location failed to produce statistically significant differences in mean time to first effect, median simulated venipuncture score, mean total sedation score and mean recovery time.