One of the challenges of current proteomics research is identifying healthy and diseased mass spectrometric (MS) patterns within biological fluids. As a result, sample preparation methodologies, as well as the mathematical tools utilized for MS data analysis become pivotal. This study involves a thorough evaluation of the reproducibility and protein resolution that various urinary protein preparation methodologies provide in MALDI MS analysis. Several precipitation approaches, ultrafiltration, as well as direct dilution of urine in MALDI MS compatible buffers were applied in combination to a thorough bioinformatics analysis of the generated MS data. Our results indicate that ultrafiltration, as well as direct dilution of urine in TFA, can provide information rich and reproducible spectra for mass ranges up to 20 kDa. The importance of the presence of peak reproducibility filters when generating disease classification models is suggested.