Real-time monitoring of the moisture content of crystal powder in a fluidized bed dryer was investigated during the conversion of lactulose trihydrate crystal powder into lactulose anhydride crystal powder. In-line near-infrared spectroscopy (NIRS) was used to measure NIR spectra of the fluidized bed drying of lactulose trihydrate crystal powder to produce lactulose anhydride crystal powder. A PLS regression model was built based on the reference moisture values after pretreatment by the MSC method. Dependencies of the spectra on dry air temperature and particle size were eliminated in the regression model. This model was externally validated by using another lot of lactulose trihydrate crystal powder in the process of fluidized bed conversion of lactulose trihydrate crystal powder into lactulose anhydride crystal powder. The predicted moisture values agreed well with the reference moisture values. The result shows that the moisture content can be estimated well by this method despite the dependencies of the NIR spectra on temperature and particle size. This confirms the feasibility of using in-line NIRS for real-time monitoring of moisture content of crystal powder in a fluidized bed dryer during the conversion of lactulose trihydrate crystal powder into lactulose anhydride crystal powder.