Elevated blood pressure is common in the emergency department, but the relationship between antihypertensive medication adherence and blood pressure in the emergency department is unclear. This cross sectional study tested the hypothesis that higher antihypertensive adherence is associated with lower systolic blood pressure in the emergency department among adults with hypertension who sought emergency department care at an academic hospital from July 2012-April 2013. Biochemical assessment of antihypertensive adherence was performed using a mass spectrometry blood assay, and the primary outcome was average emergency department systolic blood pressure. Analyses were stratified by number of prescribed antihypertensives (<3, ≥3) and adjusted for age, sex, race, insurance, literacy, numeracy, education, body mass index, and co-morbidities. Among 85 patients prescribed ≥3 antihypertensives, mean systolic blood pressure for adherent patients was 134.4 mm Hg (+/− 26.1 mm Hg), and in adjusted analysis was −20.8 mmHg (95% CI −34.2 to −7.4 mm Hg, p=0.003) different from non-adherent patients. Among 176 patients prescribed <3 antihypertensives, mean SBP was 135.5 mm Hg (+/− 20.6 mm Hg) for adherent patients, with no difference by adherence in adjusted analysis (+2.9 mm Hg, 95% CI −4.7 to 10.5 mm Hg, p=0.45). Antihypertensive non-adherence identified by biochemical assessment was common and associated with higher systolic blood pressure in the emergency department among patients who had a primary care provider and health insurance and who were prescribed ≥3 antihypertensives. Biochemical assessment of antihypertensives could help distinguish medication non-adherence from other contributors to elevated blood pressure and identify target populations for intervention.