Progressive increase of the serum pH upon standing and storage in different conditions is documented. Bilirubin albumin titration studies were performed on serum of different pH by Sephadex gel filtration and horseradish peroxidase oxidation methods. Results showed that different pH produced different titration curves and that more reproducible and consistent results were obtained with the peroxidase oxidation titration method. Addition of HC1 and CO2to the serum also produced different bilirubin‐protein titration results. For blirubin albumin binding studies, we suggest to adjust the pH to 7.4 with CO2to obtain clinically meaningful dat