Extrusion-based bioprinting (EBP) is limited by loss of pattern fidelity when printing on wet substrates. This can be overcome using aqueous two-phase systems (ATPSs) as novel ink formulations for EBP. In this study, optimal concentrations of ATPS “inks” were determined and used to pattern human epidermal keratinocyte (HEK001) colonies on a wet substrate for promoting epidermal growth. Four equilibrated and non-equilibrated ATPS formulations were tested for stable ATPS formation and uniform cell patterning. We identified an optimal formulation that produced stable droplets on a standard tissue culture plate coated with PEG. This process was also tested on an acellular dermal matrix (DermGENTM ) to evaluate biopattern fidelity on a tissue matrix. Cell proliferation and formation of adherens junctions between cells were analyzed by immunocytochemistry. Non-equilibrated 5.0% PEG and 5.0% DEX solutions formed tighter colonies than equilibrated solutions containing identical total polymer concentrations. Cells patterned in colonies displayed higher cell viability and increased formation of E-cadherin junctions compared to non-patterned cells. Finally, when the cells were patterned on DermGENTM , discrete cell colonies were observed. This suggests that ATPS EBP holds promise for biopatterning epidermal keratinocyte cells to improve skin tissue engineering.