RATE-seq is a 4-thiouracil (4-tU)-based method that enables the in vivo measurement of transcriptome-wide RNA degradation rates. 4-tU is an analog of uracil that is rapidly incorporated into newly synthesized RNA and facilitates the conjugation of a biotinylated molecule containing a reactive thiol group. The biotinylated RNA can then be fractionated from the unlabeled RNA with streptavidin magnetic beads. By adding 4-tU to a culture of cells growing in steady-state conditions, fractionating the labeled population of RNA at multiple time points following 4-tU addition, and quantifying the abundance of newly transcribed RNAs using RNAseq, it is possible to estimate the degradation rates of all transcripts in a single experiment. The analysis of the RATE-seq data entails normalization of RNAseq libraries to thiolated RNA spike-ins and nonlinear model fitting to estimate the degradation rate constant for each RNA species.