Abstract Background Reported rates of medication non-adherence in patients with heart failure (HF) vary widely, ranging between 5-60%, depending on the method of assessment. Very few studies have assessed non-adherence objectively by measuring the plasma level of medications or metabolites; the true prevalence of medication non-adherence in HF patients is unknown. Objectives To report prevalence and clinical associates of medication non-adherence in a cohort of ambulatory HF patients assessed using liquid chromatography–tandem mass spectrometry analysis of plasma levels of prescribed, evidence-based treatments. Methods We assessed adherence in 172 consecutive patients with HF attending a routine clinic follow-up visit. We used liquid chromatography-tandem mass spectrometry to measure the presence of prescribed medications (angiotensin converting enzyme inhibitors (ACEi), angiotensin receptor blocker (ARB), beta-blockers (BB), mineralocorticoid receptor antagonists (MRA), diuretics and digoxin) in plasma samples collected during the visit. No patient knew in advance that the blood test would be used to assess adherence. Results 69% were male; median (IQR) age 76 (67-82) years; median NTproBNP 1036 (433-2340) ng/L; 62% HF with reduced ejection fraction (HFrEF, LVEF <40%). 1 in 5 patients had severe symptoms (NYHA III/IV) and 38% patients had Clinical Frailty Scale ≥ 5. 120 (70%) had all prescribed medications of interest detectable in plasma; in 52 (30%) patients, at least one prescribed medication was not detected. In none of the patients were no prescribed medications detected (Figure 1). Among the 125 patients prescribed diuretics, 18 (14%) were non-adherent. Among the 106 patients with HFrEF, 25 (24%) were at least partially non-adherent to a disease modifying medication (ACEi/ ARB/BB/MRA). Amongst medication groups studied, the highest non-adherence rate was with ACEi/ARB and diuretics (14% for each) (Figure 2). Comparing non-adherent patients to those who were adherent, there was no significant difference in age, sex or number of medications prescribed. Frailty was more common in the non-adherent compared to adherent group (46% vs 34%). Systolic/ diastolic blood pressure and heart rate were higher in non-adherent patients [median (IQR) SBP: 150 (137-174) vs 140 (125-164) mmHg; median (IQR) DBP 80 (69-89) vs 75 (64-83) mmHg; median (IQR) HR 76 (67-85) vs 69 (60-77) mmHg, each p<0.05]. NTproBNP tended to be higher in those who were non-adherent [median (IQR): 1309 (673-2302) vs 891 (353-2391) ng/L]. Conclusion Medication non-adherence, assessed using a single spot plasma measurement of medication levels, appears to be common in ambulatory patients with HF. 1 in 3 patients were at least partially non adherent to prescribed medications. Our observations have implications for the assessment and management of patients with HF.