Updated project metadata. Quantitative proteomics has matured into an established tool and longitudinal proteomic experiments have begun to emerge. However, no effective, simple-to-use differential expression method for longitudinal proteomics data has been released. Typically, such data is noisy, contains missing values, has only few time points and biological replicates. To address this need, we provide a comprehensive evaluation of several existing differential expression methods for high-throughput longitudinal omics data and introduce a new method, Robust longitudinal Differential Expression (RolDE). To evaluate the longitudinal differential expression methods in a real proteomics study setting, we generated and used an experimental membrane enriched longitudinal proteomics data of Francisella tularensis subspecies novicida (Fn). For evaluation of the methods, differential expression between the null mutant and the wild type strains was investigated,while their growth at different temperatures offered a surrogate for the different time points. Technical replicates of the samples were used to form technical replicate datasets analyzed with the different methods to evaluate the reproducibility of the methods. In addition, the Fn dataset with known protein modifications, was used to explore the evaluated methods ability to detect these proteins and other closely associated pathway proteins expected to be affected.