Enzyme immobilization is a beneficial component involved in biocatalytic strategies. Understanding and evaluating the enzyme immobilization system plays an important role in the successful development and implementation of the biocatalysis route. Ensuring the implementation of a successful enzyme immobilization process is vital for realizing a highly functioning and well suited biocatalytic process within pharmaceutical development. To develop a method which can accurately and objectively identify and classify differences within enzyme immobilization systems, sample preparation methods, and data collection parameters. Raman hyperspectral imaging was used to obtain a total of eight spectral data sets from enzyme immobilization samples. Partial least squares discriminant analysis (PLS-DA) was used to classify and identify the samples based on their differences. Several two-class, four-class, and eight-class PLS-DA models were built to classify the different sample data sets. All models reached between 92-100% accuracy after cross-validation and external validation, illustrating great success of the models for identifying differences between the samples. Raman hyperspectral imaging with machine learning can be used to investigate, interpret, and classify different data collection parameters, sample preparation methods, and enzyme immobilization supports, providing crucial insight into enzyme immobilization process development. © 2023. Springer Science+Business Media, LLC, part of Springer Nature.