Total protein isolation followed by quantitation, often using a colorimetric assay such as the bicinchoninic acid (BCA) assay, is a common laboratory technique. Protein samples are compared to a curve made with dilutions of a standard such as bovine serum albumin (BSA) to determine their concentrations. Experiments were designed to determine the most reproducible and accurate method for quantifying protein concentrations of a series of experimental samples over time. The effect of freezing on diluted standards was investigated. Standards were frozen at -20°C or -80°C and serially thawed and refrozen up to three times prior to their use in a BCA assay. Thawing and refreezing the standards had no significant effect on protein concentration and the resulting standard curves. Inter-person and intra-person variability in the preparation of standards was also investigated. Protein concentration differences due to inter-person and intra-person variability were greater than protein concentration variability resulting from freezing and thawing, regardless of the freezing temperature. The most reproducible and accurate method for determining the protein concentration of extracted samples in an experimental series over time is diluting a large batch of BSA standards and freezing them at either -20°C or -80°C. Reproducibility was maintained with up to three freeze-thaws.