Purpose: Transporter expression, determined by quantitative proteomics, together with PBPK model is a promising tool to scale transporter activity from in-vitro to in-vivo (IVIVE). To implement this methodology, it is the transporter protein expression in the plasma membrane that should be quantified. Here, we used an optimized cell surface biotinylation method to quantify the plasma membrane of transporters in OATP1B1, OATP2B1, OATP1B3 or NTCP-expressing cells. OATP1B1 uptake activity was measured in the same batch of cells (CHO and MDCKII) as in the biotinylation study to determine whether plasma membrane expression or total membrane expression of OATP1B1 better correlates with its activity. Methods: The Pierce cell surface isolation kit was used. OATP1B1-expressing CHO, MDCKII or HEK293 cells, OATP2B1-expressing MDCKII cells, OATP1B3-expressing HEK293 cells, and NTCP-expressing CHO cells were incubated with 0.78 mg/mL plasma membrane impermeable biotinylation reagent, sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (EZ-Link Sulfo-NHS-SS-Biotin), for 1hr at 37°C. Biotinylation reaction was quenched with glycine (100 mM) and the cells were treated with the lysis buffer. The lysate was incubated at room temperature for 1 hr with a column containing NeutrAvidin resin. After the incubation, the column containing NeutrAvidin resin was centrifuged (1000 x g for 1 min) and the non-biotinylated fraction (containing intracellular proteins) was collected. Then, the column containing NeutrAvidin resin was incubated with the elution buffer containing dithiothreitol to elute the biotinylated fraction (containing the plasma membrane proteins). The expression of OATP1B1 or OATP2B1 or OATP1B3 or NTCP, Na+- K+ ATPase (plasma membrane marker), and calreticulin (endoplasmic reticulum marker) was quantified in the lysate, the non-biotinylated and the biotinylated fractions by quantitative proteomics (LC-MS/MS). Estradiol 17-β glucuronide ([3H]-EG) (10 nM) was used as a substrate to measure OATP1B1 transport activity in OATP1B1-expressing CHO and MDCKII cells. Results and conclusion: Mean (± SD) percent of OATP1B1 expressed in the plasma membrane of CHO, MDCKII and HEK293 cells was 79.7% (±4.7%), 67.7% (±12.2%) and 65.3 (±6.8%), respectively. The percent of plasma membrane expression of OATP1B1 in MDCKII (p<0.005) or HEK293 (p<0.007) was significantly lower (Mann–Whitney U test) than that in CHO cells. The percent of plasma membrane expression of OATP2B1 in MDCKII cells, 37.1% (±15.7%), was noticeably lower than that of OATP1B3 in HEK293 cells, 63.2% (±1.6 %), or NTCP in CHO cells, 71.7% (±1.2%). Recovery of all proteins was found to be 79.4-115.7%. Negligible presence (<12%) of the intracellular protein, calreticulin, was observed in the plasma membrane fraction. The percent of Na+- K+ ATPase in the plasma membrane closely tracked the percent of transporter expressed in the plasma membrane. OATP1B1 activity, as measured by [3H]-EG uptake, was better correlated with OATP1B1 expression in the plasma membrane (r2 = 0.96) versus in the cell (r2 = 0.5) for CHO and MDCKII cells. The improved expression-activity correlation when only plasma membrane expression was used and the low plasma membrane expression of OATP2B1 in MDCKII cells demonstrate the importance of quantifying plasma membrane expression of a transporter for meaningful IVIVE. This work was supported in part by the Simcyp Grant & Partnership Scheme.