Many components in urine are useful in clinical diagnosis and urinary proteins are known as important components to define many diseases such as proteinuria, kidney, bladder and urinary tract diseases. In this study, we focused on the comparison of different sample preparation methods for isolating urinary proteins prior to protein analysis of pooled healthy and lung cancer patient samples. Selective method was used for preliminary investigation of some putative urinary protein markers. Urine samples were passed first through a gel filtration column (PD-10 desalting column) to remove high salts and subsequently concentrated. Remaining interferences were removed by ultrafiltration or four precipitation methods. The analysis of urinary proteins by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed many similarities in profiles among preparation methods and a few profiles were different between normal and lung cancer patients. In contrast, the results of two-dimensional gel electrophoresis (2-DE) showed more distinctly different protein patterns. Our finding showed that the sequential preparation of urinary proteins by gel filtration and ultrafiltration could retain most urinary proteins which demonstrated the highest protein spots on 2-D gels and able to identify preliminary urinary protein markers related to cancer. Although sequential preparation of urine samples by gel filtration and protein precipitation resulted in low amounts of proteins on 2-D gels, high Mr proteins were easily detected. Therefore, there are alternative choices for urine sample preparation for studying the urinary proteome and identifing urinary protein markers important for further preclinical diagnostic and therapeutic applications.