BackgroundPhysicians routinely encounter diagnostic uncertainty in practice. Despite its impact on health care utilization, costs and error, measurement of diagnostic uncertainty is poorly understood. We conducted a systematic review to describe how diagnostic uncertainty is defined and measured in medical practice.MethodsWe searched OVID Medline and PsycINFO databases from inception until May 2017 using a combination of keywords and Medical Subject Headings (MeSH). Additional search strategies included manual review of references identified in the primary search, use of a topic-specific database (AHRQ-PSNet) and expert input. We specifically focused on articles that (1) defined diagnostic uncertainty; (2) conceptualized diagnostic uncertainty in terms of its sources, complexity of its attributes or strategies for managing it; or (3) attempted to measure diagnostic uncertainty.Key ResultsWe identified 123 articles for full review, none of which defined diagnostic uncertainty. Three attributes of diagnostic uncertainty were relevant for measurement: (1) it is a subjective perception experienced by the clinician; (2) it has the potential to impact diagnostic evaluation—for example, when inappropriately managed, it can lead to diagnostic delays; and (3) it is dynamic in nature, changing with time. Current methods for measuring diagnostic uncertainty in medical practice include: (1) asking clinicians about their perception of uncertainty (surveys and qualitative interviews), (2) evaluating the patient–clinician encounter (such as by reviews of medical records, transcripts of patient–clinician communication and observation), and (3) experimental techniques (patient vignette studies).ConclusionsThe term “diagnostic uncertainty” lacks a clear definition, and there is no comprehensive framework for its measurement in medical practice. Based on review findings, we propose that diagnostic uncertainty be defined as a “subjective perception of an inability to provide an accurate explanation of the patient’s health problem.” Methodological advancements in measuring diagnostic uncertainty can improve our understanding of diagnostic decision-making and inform interventions to reduce diagnostic errors and overuse of health care resources.Electronic supplementary materialThe online version of this article (10.1007/s11606-017-4164-1) contains supplementary material, which is available to authorized users.