Background Atlantoaxial (AA) cerebrospinal fluid (CSF) collection in standing horses utilizes the controlled narcotic morphine, thereby limiting feasibility in field practice settings. Objectives To compare AA CSF collection time and reaction scores in horses sedated with morphine‐containing and opioid‐free sedation protocols: detomidine + morphine (DM), detomidine + xylazine (DX), detomidine + detomidine (DD), detomidine alone (D0); To develop a novel method for assessing sedation in standing horses using open‐source motion‐tracking software. Animals Six healthy adult horses. Methods Randomized crossover. Atlantoaxial CSF collections were performed weekly for 4 weeks. Horses received sedation protocols in random order. Procedure time and procedure reaction scores were compared between groups using Friedman test. Associations between procedure reaction scores and motion tracking variables (total distance and farthest excursion traveled by horse's head) were examined using scatter diagrams and linear regression. Results Procedure times were lower in horses sedated with DX (median: 36 seconds; range: 28 − 188), compared to D0 (121; 35 − 196; P = 0.04). Procedure reaction scores were lower in horses sedated with DX (median 1.0, range 1.0 to 2.0), or DM (1.8; 1.3 − 3.0) compared to DO (3.0; 2.3 − 3.0; P = .03). Reactions to dura mater puncture were recorded in 3 of 6 horses in D0 and DD groups, and 0 of 6 horses in DX and DM groups. Positive associations were observed between reaction score vs total distance or farthest excursion distance from baseline. Conclusions and Clinical Importance Both opioid‐free and morphine‐containing sedation protocols are acceptable for AA CSF collection. Motion‐tracking software represents a novel method for assessing sedation in standing horses.