Assessment of glomerular filtration rate (GFR) is fundamental to clinical practice, public health, and research. The kidney has several critical functions; GFR is used as an overall assessment of these kidney functions. GFR is used to diagnose, stage, and manage chronic kidney disease (CKD); ascertain the prognosis for chronic kidney disease-related events and mortality; and determine drug dosages. GFR is the rate at which the glomerulus filters plasma to produce an ultrafiltrate and can be assessed from clearance or serum levels of filtration markers. Clearance measurements using exogenous filtration markers are difficult to perform in routine clinical practice, so GFR is more commonly estimated through equations based on serum concentrations of endogenous filtration markers, most commonly creatinine. These GFR estimates are reasonably accurate, but optimal care for patients may require a confirmatory test for a more accurate GFR assessment. Confirmatory tests currently available include cystatin C-based equations, urinary or plasma clearance of exogenous filtration markers, or urinary clearance of creatinine. Appreciation of the concept of GFR and methods for optimal assessment in routine practice or special circumstances, and their strengths and limitations, are critical in making judicious use of the available tools.