A stable HEK293T cell line expressing the human voltage-gated proton channel Hv1 was generated using CRISPR/Cas9-mediated knock-in at the RPL13A locus, a housekeeping gene known for consistent expression. A multicistronic cassette encoding Hv1, GFP, and a Zeocin resistance gene enabled both selection and fluorescence-based monitoring. Successful integration was confirmed by GFP expression in over 99% of cells and functional analysis using patch-clamp electrophysiology. The Hv1 current showed sensitivity to pH changes and inhibition by NZ58, confirming physiological channel behavior. This stable cell line overcomes limitations of transient expression systems and provides a reliable platform for long-term functional and pharmacological studies of Hv1.