Updated project metadata. Prokaryotic genome annotation is highly dependent on automated methods, as manual curation cannot keep up with the exponential growth of sequenced genomes. Current automated techniques depend heavily on sequence context and often underestimate the complexity of the proteome. We developed REPARATION (RibosomeE Profiling Assisted (Re-)AnnotaTION), a de novo algorithm that takes advantage of experimental evidence from ribosome profiling (Ribo-seq) to delineate translated open reading frames (ORFs) in bacteria, independent of genome annotation. Ribo-seq next generation sequencing technique that provides a genome-wide snapshot of the position translating ribosome along an mRNA at the time of the experiment. REPARATION evaluates all possible ORFs in the genome and estimates minimum thresholds to screen for spurious ORFs based on a growth curve model. We applied REPARATION to three annotated bacterial species to obtain a more comprehensive mapping of their translation landscape in support of experimental data. In all cases, we identified hundreds of novel ORFs including variants of previously annotated and novel small ORFs (<71 codons). Our predictions were supported by matching mass spectrometry (MS) proteomics data and sequence conservation analysis. REPARATION is unique in that it makes use of experimental Ribo-seq data to perform de novo ORF delineation in bacterial genomes, and thus can identify putative coding ORFs irrespective of the sequence context of the reading frame.