Multidimensional protein identification technology (MudPIT) is a powerful method for the identification of proteins from complex samples. This technique combines two-dimensional chromatography, in the form of reverse phase chromatography coupled with cation exchange chromatography, with tandem mass spectrometry to generate data that can be used to determine the proteins present in a number of complex biological samples like cells, tissues and biological fluids. MudPIT has advantages over traditional methods of protein identification because it requires fewer sample-handling steps, increases protein recovery, and can better resolve proteins that were previously difficult to recover such as very large or very small proteins, hydrophobic proteins, or proteins with extreme isoelectric points. Our current efforts focus on the determination of proteins present in mouse urine, specifically those that would elicit an olfactory response. A number of factors must be refined in order to maximize the number of detected olfactory proteins. These factors include initial sample volume and concentration, sample preparation, chromatographic gradients. To this end, we have analyzed a number of urine samples from male and female mice in various reproductive states to resolve all of the previously mentioned factors and determine the optimal analytical conditions. This presentation will focus on the methodology used in the determination of our current analytical conditions.