Pyrolysis oil is a renewable resource derived from biomass and can be viewed as a potential crude oil substitute or extender. Pyrolysis oil has several undesirable properties, including very high acid and water contents. Because of the high acid content, pyrolysis oil will likely be blended with conventional crude oil for processing into transportation fuel, lowering the overall acid content of the mixture and mitigating the corrosivity toward refining units. Pyrolysis oil contains polar and nonpolar organics, water, and particulates. These components can be difficult to homogenize and, if left standing, may separate again. Additionally, pyrolysis oil will thicken with time as components react to form higher molecular weight products. ASTM D664 Standard Test Method for Acid Number of Petroleum Products by Potentiometric Titration is often used for determining the acid content of pyrolysis oils. However, ASTM D664 was developed specifically for petroleum-based lubricants. The ASTM D664 solvent system is unsuited for use with pyrolysis oil, failing to completely solubilize the pyrolysis oil and yielding unreliable results. This work addresses two related problems with pyrolysis oil. The first is obtaining and maintaining a stable, homogeneous sample for analysis that is representative of the material to be analyzed. The second issue is the development of a reliable method for the measurement of the acid concentration by titration, which is necessary to calculate the amount of pyrolysis oil that can be mixed with a crude oil and safely processed in existing refinery equipment. A simple solvent system based on ethylene glycol (ethane-1,2-diol) has been devised that produces stable, homogeneous solutions and allows for highly repeatable acid numbers on pyrolysis oil. The accuracy and precision for the modified method are both satisfactory.