The technique equilibrium sampling through membrane (ESTM) was extended to measuring the free drug concentration in solutions of drug and protein. Bjerrum and Scatchard plots were employed for characterizing individual drug binding to pure human blood proteins. Four drugs were investigated as a model system: fluvoxamine and ropivacaine which dominantly bind to alpha-acid glycoprotein (AGP), and R,S-ibuprofen and S-ketoprofen which highly bind to human serum albumin (HSA). The level of drug binding to AGP and HSA relied on drug and protein concentrations. Bjerrum and Scatchard plots revealed high affinity constants (Ka) at low protein concentration. Both Bjerrum and Scatchard plots of fluvoxamine and ropivacaine binding to AGP showed one specific binding site (n1=1) with ropivacaine Ka value close to 5 times higher than the Ka of fluvoxamine at 22.9 microM AGP concentration. Bjerrum plots of ketoprofen and ibuprofen gave total number of binding sites or bound molecules of 6-7, which did not depend on the drug or protein concentration. Scatchard plots of ketoprofen and ibuprofen exhibited two binding sites (n1 and n2) at 0.15 microM and 0.75 microM HSA concentrations. On one hand, at 0.15 microM HSA, ketoprofen and ibuprofen were bound to site I at n1=1.2 and n1=1.0, respectively. However, at 0.75 microM HSA, ketoprofen and ibuprofen were bound to site I at n1=1.2 and n1=1.9, respectively. On the other hand, site II, at 0.15 microM HSA, interacted with ketoprofen and ibuprofen at n2=5.6 and 6.7, respectively. However, at 0.75 microM HSA, site II interacted with ketoprofen at n2=7.4 and ibuprofen at n2=6.2. It would be concluded that, upon mixing ketoprofen and ibuprofen in a HSA solution, a ketoprofen-ibuprofen interaction would most likely occur at site II in HSA.