Finding a long-term, human-relevant culture model for primary human hepatocytes (PHHs) for pharmacological and toxicological studies remains a challenge. Current in vitro model platforms are often inconvenient and complex, lack phenotypic stability over time, and do not support multiple PHH lots, lacking experimental reproducibility and flexibility. Here, we provide a detailed protocol for the thawing, plating, and maintenance of an all-human 2D+ hepatic system (TV2D+), which takes advantage of standard two-dimensional (2D) culture techniques and equipment while maintaining the longevity and phenotypic stability over time that typically accompany more complex three-dimensional (3D) systems. The results show attachment and percent plateability in TV2D+ as a function of PHH seeding density, as well as stable functionality for at least 2 weeks in culture. A range of PHH seeding densities are assessed to achieve a successful long-term culture. When established properly, the PHHs in TV2D+ organize into hepatocyte colonies, express a hepatic-specific marker, and maintain viability, architectural integrity, and physiologically relevant levels of albumin and urea. This unique combination of attributes makes the TV2D+ system a suitable hepatic model for a variety of pharmacological and toxicological applications.