5-Hydroxymethylcytosine (5hmC) is a modified form of cytosine, which has recently been found in mammalian cells and tissues. 5hmC is derived from 5-methylcytosine (5mC) by Ten-eleven translocation (TET) family protein-mediated oxidation and may regulate gene expression. Numerous affinity-based profiling methods have been developed to help understand the exact function of 5hmC in the genome. However, these methods have a relatively low resolution (~100 bp) without quantitative information of the modification percentage on each site. Here we demonstrated the detailed procedure of Tet-Assistant Bisulfite Sequencing (TAB-Seq), which can detect 5hmC at single-base resolution and quantify its abundance at each site. In this protocol, the genomic DNA is first treated with βGT and recombinant mTet1 consecutively to convert 5hmC to 5gmC and 5mC to 5caC, respectively. The treated genomic DNA can be directly applied to bisulfite treatment to detect 5hmC on specific loci or applied to whole-genome bisulfite sequencing as needed.