Evidence-based guidelines in HIV care aim to improve patients’ health outcomes, quality of care, and cost-effectiveness. Laboratory monitoring plays an important role in assessing clinical status of patients and forms an integral part of HIV treatment guidelines. The Dutch HIV monitoring foundation (Stichting HIV Monitoring) previously observed variation between HIV treatment centres in the Netherlands in terms of compliance with guidelines for performing laboratory tests. Drawing on qualitative research methods, this article aimswe aimed to describe factors that influence guideline compliance for laboratory monitoring in outpatient HIV care in the Netherlands. Twelve semi-structured in-depth interviews were conducted with a convenience sample of physicians from four HIV treatment centres. In general, physicians perceived laboratory guidelines as useful. However, unclear online visual representation of the guidelines, a lack of set reminders for tests, and assessment of patients’ risk behaviour, which differs per patient, were identified as barriers to guideline compliance. The compartmentalisation of the Dutch healthcare system was viewed as hampering guideline compliance. A clinical-decision-support tool could possibly facilitate compliance with laboratory monitoring guidelines. Moreover, better alignment of HIV outpatient care, municipal health services and primary care, in terms of laboratory testing, could optimize efficiency, increase cost-effectiveness, and improve quality of HIV care.