Alignments in bioinformatics refer to the arrangement of sequences to identify regions of similarity that can indicate functional, structural, or evolutionary relationships. They are crucial for bioinformaticians as they enable accurate predictions and analyses in various applications, including protein subcellular localization. The predictive model used in this article is based on a deep – convolutional architecture. We tested configurations of Deep N‐to‐1 convolutional neural networks of various depths and widths during experimentation for the evaluation of better‐performing values across a diverse set of eight classes. For without alignment assessment, sequences are encoded using one‐hot encoding, converting each character into a numerical representation, which is straightforward for non‐numerical data and useful for machine learning models. For with alignments assessment, multiple sequence alignments (MSAs) are created using PSI‐BLAST, capturing evolutionary information by calculating frequencies of residues and gaps. The average difference in peak performance between models with alignments and without alignments is approximately 15.82%. The average difference in the highest accuracy achieved with alignments compared with without alignments is approximately 15.16%. Thus, extensive experimentation indicates that higher alignment accuracy implies a more reliable model and improved prediction accuracy, which can be trusted to deliver consistent performance across different layers and classes of subcellular localization predictions. This research provides valuable insights into prediction accuracies with and without alignments, offering bioinformaticians an effective tool for better understanding while potentially reducing the need for extensive experimental validations. The source code and datasets are available at http://distilldeep.ucd.ie/SCL8/.