Updated FTP location. We developed a simple and effective method for “in-depth” membrane proteomics. We optimize the sample preparation for the maximum numbers of the identified membrane proteins using a human cell line, HEK293T, as a model. From this study, we demystified the merits of Urea wash methods for membrane enrichment. The membrane enrichment by Urea Wash was elucidated and compared to Alkaline Wash and without washing. The optimized protocol provides better comprehensiveness of the membrane proteome and enhances data quality.