The global COVID-19 pandemic, caused by SARS-CoV-2, has increased scientific interest in coronaviruses. Controlling environmental conditions is crucial to prevent viral transmission via surfaces or air. This study aimed to evaluate the effect of storage time and heat on the stability of human coronavirus-229E (HCoV-229E) using Raman spectroscopy. Wild-type and GFP-tagged HCoV-229E were stored at −20 °C for 24, 168, and 504 h, and exposed to 37 °C for 120 min, and 95 °C for 10 min. Raman spectra were collected at 532 nm excitation (3 s exposure, 7 mW). Multivariate analyses (PCA, OPLS-DA) distinguished virus types, concentrations, and environmental effects, identifying structural changes linked to stress conditions. Continuous OPLS-DA models explained 97% of variance (R 2Y) and predicted 95% (Q 2), demonstrating Raman spectroscopy’s potential for detecting molecular changes in coronaviruses under varying conditions.