Protein microarrays are miniaturized two-dimensional arrays, incorporating thousands of immobilized proteins, typically printed in minute amounts on functionalized solid substrates, which can be analyzed in a high-throughput fashion. Irreproducibility of the printing techniques adopted, resulting in inconsistently and nonuniformly deposited microscopic spots, nonuniform signal intensities from the printed microspots, and significantly high background noise are some of the critical issues that affect protein analysis using traditional protein microarrays. To overcome such issues, in this study, we introduced a novel gold grid pattern-based protein microarray. The grid patterns incorporated in our microarray are equivalent to the spots used for protein analysis in conventional protein microarrays. We utilized the signal intensities from the grid patterns acting as spots for quantifying the protein concentration levels. To demonstrate the utility of our novel design concept, we quantified as low as 66.7 ng/mL of bovine serum albumin using our gold grid pattern-based protein microarray. Our grid pattern-based design concept for protein quantification overcame the signal nonuniformity issues and ensured that the dominance of any distorted signal from a single spot did not affect the overall protein quantification results as encountered in conventional protein microarrays.