Non-alcoholic fatty liver disease (NAFLD) has become a global pandemic. However, a pharmacological cure has not been approved for NAFLD treatment. The greatest barriers to the development of new treatments are the ambiguous criteria among the NAFLD stages and the lack of quantitative methodologies for its disease assessment in a translatable preclinical model. In this study, we developed impedance assessment systems to quantify NAFLD progression in three-dimensional (3D) liver microtissue (hMT). The hMT model undergoing NAFLD represents clinical-like characteristics for a range of stages, such as lipid accumulation, cell ballooning, and stiffening. Each stage can be quantitatively assessed by an impedance system with microchannels under constant or dynamic pressure, depending on the relevant mechanical and morphological changes used in the clinical assessment of NAFLD. We determined a correlation between the impedance parameters and pathophysiological characteristics, such as gap widening and cytoplasmic deformation associated with NAFLD progression using bioimpedance simulation, showing hMTs struggling to return to normal states. In addition, we identified the relative stiffness to assess fibrogenesis from the correlation of resistance change and elongation length into the smaller channel of hMTs. We hope this methodology will have a significant impact on drug development by facilitating improved NAFLD assessment.