Evaluation of freeze-dried biopharmaceutical formulations requires careful analysis of multiple quality attributes. The aim of this study was to evaluate the use of near-infrared (NIR) imaging for fast analysis of water content and related physical properties in freeze-dried formulations. Model formulations were freeze-dried in well plates. Samples were imaged with a NIR hyperspectral camera after freeze-drying and upon storage. On the basis of Karl Fischer titration reference values, a univariate quantification model was constructed and used to visualize the distribution of water within freeze-dried samples. Differences observed between samples stored at 11% and 43% relative humidity (RH) were found to be related to the amount of amorphous component in the sample. When stored at 43% RH, the moisture content in samples with high sucrose content increased within 2 days and some degree of localized drying was observed within the samples after 3 days of storage. Further investigations with X-ray powder diffraction confirmed this local drying to be related to crystallization of sucrose. The combination of fast analysis of water content and spatial solid-state information makes NIR imaging a powerful tool for formulation development of freeze-dried samples. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.