PurposeOptimization and validation of a sequence for measuring the labeling efficiency of pseudocontinuous arterial spin labeling (pCASL) perfusion MRI.MethodsThe proposed sequence consists of a labeling module and a single slice Look‐Locker echo planar imaging readout. A model‐based algorithm was used to calculate labeling efficiency from the signal acquired from the main brain‐feeding arteries. Stability of the labeling efficiency measurement was evaluated with regard to the use of cardiac triggering, flow compensation and vein signal suppression. Accuracy of the measurement was assessed by comparing the measured labeling efficiency to mean brain pCASL signal intensity over a wide range of flip angles as applied in the pCASL labeling.ResultsSimulations show that the proposed algorithm can effectively calculate labeling efficiency when correcting for T1 relaxation of the blood spins. Use of cardiac triggering and vein signal suppression improved stability of the labeling efficiency measurement, while flow compensation resulted in little improvement. The measured labeling efficiency was found to be linearly (R = 0.973; P < 0.001) related to brain pCASL signal intensity over a wide range of pCASL flip angles.ConclusionThe optimized labeling efficiency sequence provides robust artery‐specific labeling efficiency measurement within a short acquisition time (∼30 s), thereby enabling improved accuracy of pCASL CBF quantification. Magn Reson Med 77:1841–1852, 2017. © 2016 International Society for Magnetic Resonance in Medicine Magn Reson Med 77:1841–1852, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.