Pasteurized juices may undergo spoilage during normal shelf life due to Alicyclobacillus spp. Metabolic byproducts during germination of these thermoacidiophilic, endospore-forming bacteria impart off-flavors. The objective was to develop a simple, rapid, and sensitive approach for differentiation of Alicyclobacillus spp. by attenuated total reflectance infrared (ATR-IR) microspectroscopy after isolation onto hydrophobic grid membrane (HGM) filters. Dilutions of four different species of Alicyclobacillus were filtered onto HGM, incubated on orange serum agar (50 °C, 36−48 h), and dried under vacuum. Spectra were collected using ATR-IR microspectroscopy and analyzed by multivariate analysis. Results indicated that soft independent modeling of class analogy models exhibited clusters that permitted classification at species and strain levels. The methodology was validated by correctly predicting Alicyclobacillus (100%) in blind tests. The proposed procedure permits chemically based classification of intact microbial cells. Implementation provides the juice industry with a rapid screening procedure to detect and monitor Alicyclobacillus that threatens the quality of pasteurized juices.