VaxiJen is an online server for predicting protective antigens, tumour antigens, and subunit vaccines without relying on sequence alignment. The server uses an alignment-free approach based on auto cross covariance (ACC) transformation of protein sequences into uniform vectors of principal amino acid properties. This method allows for the prediction of antigenicity based on physicochemical properties rather than sequence similarity. The study used three datasets: bacterial, viral, and tumour proteins. Each dataset included 100 known antigens and 100 non-antigens. Models were developed using these datasets and validated through internal leave-one-out cross-validation and external validation. The models achieved prediction accuracies ranging from 70% to 89%. The server, named VaxiJen, is freely available online at http://www.jenner.ac.uk/VaxiJen. VaxiJen can be used independently or in combination with alignment-based prediction methods. It is particularly useful for identifying antigens that may not have obvious sequence similarity to known antigens. The server provides results including the predicted probability of a protein being a protective antigen or non-antigen, based on a predefined cutoff. The z descriptors, derived from principal component analysis of 29 molecular descriptors, represent the main physicochemical properties of amino acids. These descriptors are used in conjunction with ACC transformations to enhance the class-discriminating properties of the data. The ACC transformation helps to remove irrelevant information such as sequence length and amplify the class-discriminating properties of the data. The server is designed to handle single proteins or whole proteomes submitted in FASTA format. It provides results for each protein, indicating whether it is likely to be a protective antigen or non-antigen. The server is an open system, with the potential for future inclusion of models for parasite and fungal antigens. VaxiJen is the first server for alignment-independent prediction of protective antigens. It is a reliable and consistent tool for the prediction of protective antigens, and can be used in conjunction with other bioinformatics tools for reverse vaccinology. The server is freely available and can be accessed online.VaxiJen is an online server for predicting protective antigens, tumour antigens, and subunit vaccines without relying on sequence alignment. The server uses an alignment-free approach based on auto cross covariance (ACC) transformation of protein sequences into uniform vectors of principal amino acid properties. This method allows for the prediction of antigenicity based on physicochemical properties rather than sequence similarity. The study used three datasets: bacterial, viral, and tumour proteins. Each dataset included 100 known antigens and 100 non-antigens. Models were developed using these datasets and validated through internal leave-one-out cross-validation and external validation. The models achieved prediction accuracies ranging from 70% to 89%. The server, named VaxiJen, is freely available online at http://www.jenner.ac.uk/VaxiJen. VaxiJen can be used independently or in combination with alignment-based prediction methods. It is particularly useful for identifying antigens that may not have obvious sequence similarity to known antigens. The server provides results including the predicted probability of a protein being a protective antigen or non-antigen, based on a predefined cutoff. The z descriptors, derived from principal component analysis of 29 molecular descriptors, represent the main physicochemical properties of amino acids. These descriptors are used in conjunction with ACC transformations to enhance the class-discriminating properties of the data. The ACC transformation helps to remove irrelevant information such as sequence length and amplify the class-discriminating properties of the data. The server is designed to handle single proteins or whole proteomes submitted in FASTA format. It provides results for each protein, indicating whether it is likely to be a protective antigen or non-antigen. The server is an open system, with the potential for future inclusion of models for parasite and fungal antigens. VaxiJen is the first server for alignment-independent prediction of protective antigens. It is a reliable and consistent tool for the prediction of protective antigens, and can be used in conjunction with other bioinformatics tools for reverse vaccinology. The server is freely available and can be accessed online.