•A method is presented for 5hmC detection and analysis using Infinium 450K BeadChips.•The oxBS-450K method can discriminate between 5mC and 5hmC in human gDNA•5hmC levels were quantified genome-wide in 3 distinct biological samples.•The reported 5hmC signal was validated using mass spectrometry and pyrosequencing.•The effects of differing amounts of input DNA on final 5hmC call rate are discussed. DNA methylation analysis has become an integral part of biomedical research. For high-throughput applications such as epigenome-wide association studies, the Infinium HumanMethylation450 (450K) BeadChip is currently the platform of choice. However, BeadChip processing relies on traditional bisulfite (BS) based protocols which cannot discriminate between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Here, we report the adaptation of the recently developed oxidative bisulfite (oxBS) chemistry to specifically detect both 5mC and 5hmC in a single workflow using 450K BeadChips, termed oxBS-450K. Supported by validation using mass spectrometry and pyrosequencing, we demonstrate reproducible (R2>0.99) detection of 5hmC in human brain tissue using the optimised oxBS-450K protocol described here.