The emergence of CRISPR/Cas system allows for precise and efficient alteration of genetic sequences in almost any kind of living cells. Several studies have attempted to modify human hepatocytes with plasmids that encodes immortalized genes, such as hepatocytes derived from stem cells or cell lines produced by an organism. Most strategies are time consuming and requires high technical expertise, especially for the use of drug metabolism studies. This study shows that there is a possibility to develop a hepatocyte cell line for functional expression studies with modifiable CYP2C9 expression. Two CRISPR/Cas9 systems were successfully assembled to specifically target the CYP2C9 gene and reduce the expression down. The efficiency of this method was validated using standardised experimental guidelines, to confirm target selection, cleavage efficiency and an analysis of target activity. Both systems managed to reduce CYP2C9 expression by at least 5 folds in Huh-7 cell line and 2 folds in HepG2 cell line. The successful knocking down of CYP2C9 regulation may open a path to a cheaper, time saving, and modifiable hepatocyte drug metabolism assays that can be grown in a lab.