With the high mortality rate associated with invasive fungal infections, methods for timely detection and diagnosis are necessary for appropriate and effective treatment. Testing for 1,3-β-D-glucan, a cell wall component of many medically important fungi, can be a useful adjunct in diagnosing such infections. The Fungitell assay (Associates of Cape Cod, East Falmouth, Massachusetts) is a US Food and Drug Administration-approved laboratory test that quantitatively measures 1,3-β-D-glucan levels and is widely available for clinical use as a relatively noninvasive method to aid in detecting the presence of invasive fungal infections. Numerous studies have evaluated its performance in clinical settings, and results have, overall, been favorable. It is not without its drawbacks, however, and the test must be interpreted and applied with care. Ordering practices are also widely variable among clinicians, and official guidelines have not been readily available. We present the details of this test and aim to propose evidence-based guidance for its use.