Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early and late replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and sub-nuclear position. Moreover, RT is regulated during development and is altered in disease . Exploring mechanisms linking RT to other cellular processes in normal and diseased cells will be facilitated by rapid and robust methods with which to measure RT genome wide. Here, we describe a protocol to analyse genome-wide RT by next-generation sequencing (NGS). This protocol yields highly reproducible results across laboratories and platforms. We also provide the computational pipelines for analysis, parsing phased genomes using single nucleotide polymorphisms (SNP) for analyzing imprinted RT, and for direct comparison to Repli-chip data obtained by analyzing nascent DNA by microarrays.