Abstract The protocol describes a targeted methylation library preparation upstream short read sequencing with an Illumina instrument. The protocol includes the New England Biolabs Next Enzymatic Methyl-seq Library Preparation workflow combined with the Twist Bioscience Targeted Methylation Sequencing workflow. The protocol is divided into 8 steps: fragmentation, library preparation, enzymatic conversion, indexing, pooling, hybridization, capture and amplification. Main advantages are (i) the limitation of DNA degradation using enzymatic conversion, (ii) both the specificity and efficiency of the capture especially in CpG highly rich regions although this step is the critical step due to the temperature control, and (iii) the pooling of samples into 8-plex reducing handling time and experimental costs. However, the workflow takes three working days with several break points. This protocol can be performed in standard molecular biology laboratories and it’s suitable for 96 samples. This approach can be adapted to any interesting regions using a custom panel for agronomic species and model organisms but also in human as a diagnostic tool.