Whole-transcriptome deep sequencing of RNA (RNA-Seq) is now the preferred method for transcription profiling, discovery of novel coding and non-coding transcripts, and identification of alternative splicing sites. For optimal RNA-Seq results, removal of the ribosomal RNA (rRNA) from an RNA sample is required prior to preparation of the RNA-Seq library. Oligo(dT)-based poly(A)+ RNA enrichment and oligonucleotide-based rRNA hybridization-capture are the most popular methods currently used to remove rRNA. However, even after multiple rounds of treatment, these procedures can yield an RNA preparation that still contains an unacceptably high proportion of rRNA contamination, especially when using compromised or partially degraded RNA samples. Additionally, poly(A)+ RNA enrichment results in the loss of non-coding RNAs that are an important component of the transcriptome and an area of active research. We describe a robust rRNA removal procedure (Ribo-Zero™ rRNA Removal Kits) that utilizes a greatly improved hybridization-capture process. A single Ribo-Zero reaction removes >99% of the cytoplasmic (nuclear-encoded) rRNA, as well as mitochondrial and chloroplast rRNA, from both intact and partially degraded RNA samples.