Pregnancy alters the disposition and exposure to multiple drugs indicated for pregnancy-related complications. Previous in vitro studies have shown that pregnancy-related hormones (PRHs) alter the expression and function of certain cytochrome P450s (CYPs) in human hepatocytes. However, the impact of PRHs on hepatic concentrations of non-CYP drug-metabolizing enzymes (DMEs) and transport proteins remain largely unknown. In this study, sandwich-cultured human hepatocytes (SCHH) from five female donors were exposed to vehicle or PRHs (estrone, estradiol, estriol, progesterone, cortisol, and placental growth hormone), administered individually or in combination, across a range of physiologically relevant PRH concentrations for 72 h. Absolute concentrations of 33 hepatic non-CYP DMEs and transport proteins were quantified in SCHH membrane fractions using a quantitative targeted absolute proteomics (QTAP) isotope dilution nanoLC-MS/MS method. The data revealed that PRHs altered the absolute protein concentration of various DMEs and transporters in a concentration-, isoform-, and hepatocyte donor-dependent manner. Overall, eight of 33 (24%) proteins exhibited a significant PRH-evoked net change in absolute protein concentration relative to vehicle control (ANOVA p < 0.05) across hepatocyte donors: 1/11 UGTs (9%; UGT1A4), 4/6 other DMEs (67%; CES1, CES2, FMO5, POR), and 3/16 transport proteins (19%; OAT2, OCT3, P-GP). An additional 8 (24%) proteins (UGT1A1, UGT2B4, UGT2B10, FMO3, OCT1, MRP2, MRP3, ENT1) exhibited significant PRH alterations in absolute protein concentration within at least two individual hepatocyte donors. In contrast, 17 (52%) proteins exhibited no discernable impact by PRHs either within or across hepatocyte donors. Collectively, these results provide the first comprehensive quantitative proteomic evaluation of PRH effects on non-CYP DMEs and transport proteins in SCHH and offer mechanistic insight into the altered disposition of drug substrates cleared by these pathways during pregnancy.