Western blot (WB) analysis is widely used, but obtaining consistent results can be problematic, especially when using multiple gels. This study examines WB performance by explicitly applying a method commonly used to test analytical instrumentation. Test samples were lysates from RAW 264.7 murine macrophages treated with LPS to activate MAPK and NF-kB signaling targets.Samples from the pooled cell lysates placed in every lane on multiple gels were analyzed by WBs for levels of p-ERK, ERK, IkBβ and non-target protein. Different normalization methods and sample groupings were applied to the density values and the resulting coefficients of variation (CV) and ratios of maximal to minimal values (Max/Min) were compared. Ideally with identical sample replicates the CVs would be 0 and the Max/Min 1; deviation indicating introduction of variability by the WB process. Common normalizations to reduce analytical variance, total lane protein, % Control, and p-ERK/ERK ratios, did not have the lowest CVs or Max/Min values. Normalization using the sum of target protein values combined with analytical replication most effectively reduced variability, resulting CV and Max/Min values as low as 5–10% and 1.1. These methods should allow reliable interpretation of complex experiments that require samples to be placed on multiple gels.