Antibody microarrays are a high throughput technology used to concurrently screen for protein expression. Most antibody arrays currently used are based on the ELISA sandwich approach that uses two antibodies to screen for the expression of a limited number of proteins. Also, since antigen-antibody interactions are concentration dependent, antibody microarrays need to normalize the amount of antibody that is used. In response to the limitations with the currently existing technology we have developed a single antibody based microarray where the quantity of antibody spotted is used to standardize the antigen concentration. In addition, this new array utilizes an internally controlled system where one color represents the amount of antibody spotted and the other the amount of the antigen that is used to quantify the level of protein expression. When compared to median florescent intensity (MFI) alone, normalization for antibody spot intensity decreased variability and lowered the limits of detection. This new antibody array was tested using standard cytokine proteins and also cell lysates obtained from mouse macrophages stimulated in vitro and evaluated for the expression of the cytokine proteins Interleukin (IL)-1, IL-5, IL-6, macrophage inflammatory protein (MIP)-1 and MIP-1. The levels of protein expression seen with the antibody microarray was compared with that obtained with western blot analysis and the magnitude of protein expression observed was similar with both technologies with the antibody array actually showing a greater degree of sensitivity. In summary, we have developed a new type of antibody microarray to screen for protein expression that utilizes a single antibody and controls for the amount of antibody spotted. This type of array appears at least as sensitive as western blot analysis and the technology can be scaled up for high throughput screening for hundreds of proteins in complex biofluids such as blood.