This research evaluates a freeze-dried live, attenuated virus vaccine during an accelerated stability study using Near Infrared (NIR) and Fourier Transform Infrared (FTIR) spectroscopy in addition to the traditional quality tests (i.e., potency assay and residual moisture analysis) and Modulated Differential Scanning Calorimetry (MDSC). Therefore, freeze-dried live, attenuated virus vaccines were stored during four weeks at 4 °C (i.e., recommended storage condition) and at 37 °C (i.e., accelerated storage condition) and weekly analyzed using these techniques. The potency assay showed that the virus titer decreased in two phases when the samples were stored at 37 °C. The highest titer loss occurred during the first week storage at 37 °C after which the degradation rate decreased. Both the residual moisture content and the relaxation enthalpy also increased according to this two-phase pattern during storage at 37 °C. In order to evaluate the virus and its interaction with the amorphous stabilizer in the formulation (trehalose), the NIR spectra were analyzed via principal component analysis (PCA) using the amide A/II band (5029–4690 cm−1). The FTIR spectra were also analyzed via PCA using the amide III spectral range (1350–1200 cm−1). Analysis of the amide A/II band in the NIR spectra revealed that the titer decrease during storage was probably linked to a change of the hydrogen bonds (i.e., interaction) between the virus proteins and the amorphous trehalose. Analyzing the amide III band (FTIR spectra) showed that the virus destabilization was coupled to a decrease of the coated proteins β turn and an increase of α helix. During storage at 4 °C, the titer remained constant, no enthalpic relaxation was observed and neither the Amide A/II band (NIR spectra) nor the Amide III band (FTIR spectra) varied.