Agaricus bisporus, an edible macroscopic fungus, is the most widely cultivated mushroom in the world. A. bisporus is highly susceptible to microbial attacks when stored in the refrigerator at 0 °C for more than 5 days. Hence, it is imperative to measure the freshness level of A. bisporus during its refrigeration to prevent any wastage of harvest. Herein, we propose a novel analytical technique of using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy as a non-invasive inspection tool to assess the moisture content of A. bisporus samples. The score plot of Principal Component Analysis (PCA) shows that the proposed analytical method can effectively discriminate aged and fresh A. bisporus. Finally, the application of ATR-FTIR coupled PCA for determination of storage life of fresh A. bisporus is demonstrated. Non-destructive analysis, rapidity, simplicity and high accuracy are the attributes of this inspection tool that may popularize this versatile analytical technique for the rapid determination of storage life of fresh A. bisporus during cold storage. © 2019 Elsevier B.V.