Background: Equine trigeminal-mediated (TGM) headshaking (HS) is a facial pain syndrome characterised by varying intensity and frequencies of head movements and nasal irritation. An accurate method for quantification and/or characterisation of severity is lacking. Objectives: To develop and validate an objective measure of TGMHS. Study design: Prospective case control study. Methods: Horses presenting for HS were recruited alongside those presenting for forelimb lameness (LAME) and pre-purchase examination as well as healthy controls (CONTROL). Head movement data were collected for five minutes whilst trotting on the lunge using a tri-axial accelerometer (range ±16g, sampling rate of 800Hz) on the bridle headpiece. Recordings were exported and peak detection performed using minimum and maximum thresholds of -1g and +1g (corrected for gravity) and a minimum peak width of 10 samples. Results: Fifty-six horses were included in the study; 18 TGMHS, 10 non-TGMHS, 12 LAME and 16 CONTROL. Characteristics and frequency of vertical (Z axis) head movements of TGMHS horses differed significantly from other horses. TGMHS horses had peaks with greater mean and maximum positive g-force (P<0.005) and lower mean and minimum negative g-force (P<0.001), greater frequency of peaks/min (P<0.001), and over twelve times greater percentage of peaks >+2g compared to other horses (P<0.001). Receiver operator curve characteristics of percentage of peaks >+2g (CI 0.72-0.95), percentage of peaks <-2g (CI 0.66-0.92), and percentage of peaks <-2g and >+2g (CI 0.72-0.96) showed excellent discrimination of TGMHS horses from LAME, CONTROL and non-TGMHS horses. Main limitations: Referral population of horses, small sample size, and control horses were not evaluated for orthopaedic disease. Conclusions: Accelerometer data from trotting exercise on the lunge gives an objective measure of HS and can differentiate between TGMHS, non-TGMHS, normal head movements, and those associated with forelimb lameness. Accelerometer use may aid HS diagnosis and monitoring of management strategies.