Replication timing profiles are cell type-specific and reflect genome organization changes during differentiation. In this protocol, we describe how to analyze genome-wide replication timing (RT) in mammalian cells. Asynchronously cycling cells are pulse labeled with the nucleotide analog 5-bromo-2-deoxyuridine (BrdU) and sorted into S-phase fractions on the basis of DNA content using flow cytometry. BrdU-labeled DNA from each fraction is immunoprecipitated, amplified, differentially labeled and co-hybridized to a whole-genome comparative genomic hybridization microarray, which is currently more cost effective than high-throughput sequencing and equally capable of resolving features at the biologically relevant level of tens to hundreds of kilobases. We also present a guide to analyzing the resulting data sets based on methods we use routinely. Subjects include normalization, scaling and data quality measures, LOESS (local polynomial) smoothing of RT values, segmentation of data into domains and assignment of timing values to gene promoters. Finally, we cover clustering methods and means to relate changes in the replication program to gene expression and other genetic and epigenetic data sets. Some experience with R or similar programming languages is assumed. All together, the protocol takes ∼3 weeks per batch of samples.