Near-infrared spectroscopic (NIRS) methods were developed to determine moisture in freeze-dried drug products with gas chromatography as the reference measurement. The gas chromatography method was optimized to provide a relative standard deviation of 1.1%. Because NIRS calibration models are limited by the quality of the reference measurement, all aspects of the GC technique were studied, including water standard preparation, operating conditions, and sample treatment. Vials of a freeze-dried drug product were prepared with water contents from 0.1 to 5.7% (w/w). NIR spectra were collected over the spectral range of 1100-2500 nm for these vials. Calibration models were established from these spectra by PLS regression analysis. Second-derivative spectra were used and spectral range and number of PLS factors were optimized for the lowest standard error of prediction (SEP). The best calibration was built with second-derivative spectra in the spectral range of 1850-1960 nm with three PLS factors and provided a SEP of 0.07% water (w/w). These results indicate that GC is a competitive reference method and that NIRS can be used for water determination in cases where other moisture analysis techniques are not compatible.Many methods have been developed for water determination, including Karl Fischer titration (KF), loss on drying, and chromatographic procedures. [1][2][3][4][5] However, each has pros and cons in terms of accuracy, speed, ease of operation, and compatibility with certain analytes. For example, although Karl Fischer titration is widely used, it consumes the sample and exposes the operator to toxic reagents. It also takes several minutes to complete one assay with no commonly available autosampler. Some analytes are not compatible with KF or loss on drying techniques because they react with the titrant, are volatile, or degrade. In addition, all methods are susceptible to ambient moisture if strict precautions are not taken. To determine water in a wide variety of sample types, it is necessary to consider each of the techniques that are available.