From a pharmaceutical standpoint, research on protein–protein interactions (PPIs) has grown in significance, particularly in high-concentration solutions. However, the majority of analytical techniques for researching protein interactions depend on finding nonideality in solutions that are moderately diluted (less than 50 mg/mL). Here, we describe the use of variablepathlength ultraviolet (UV)–visible absorption spectroscopy on a number of typical proteins to investigate and clarify such interactions over a broad concentration range (5–240 mg/mL). Delta absorbance (Abs), the difference between the measured absorbance and the corresponding theoretical absorbance (calculated from gravimetric dilution), was used in this study to track the change in UV absorption, also known as the extinction coefficient, over a wide range of protein concentrations. It was discovered that for three model proteins (bovine serum albumin, lysozyme, and monoclonal antibody), the Abs increased with protein content after being compensated for light scattering. We looked at the relationship between Abs readings and viscosity as a function of protein content because PPIs affect solution viscosity. Though to varying degrees for various proteins, the magnitude of Abs and solution viscosity followed comparable trends with increasing protein content. These findings bolster the application of such Abs measurements as a substitute method for tracking and assessing interactions in protein solutions at high concentrations. Copyright 2012 The American Pharmacists Association and Wiley Periodicals, Inc. 2012; J Pharm Sci 101:3051–3061