The integration of proteomics to other omics technologies and generation of proteome maps of a particular cell/tissue requires the identification and quantification of a maximum number of proteins. Traditional 2-D gel-based approach though provides a clear proteome map has its limitations, such as time consuming, requiring high skill, and most importantly, inability to identify low-abundance proteins. The most common drawback of 2-D gel electrophoresis is the masking of low amount proteins by the highly expressed (high abundance) proteins. Therefore, the elucidation of complete regulatory networks of a cell/tissue demands identification of low-abundance proteins. Low-abundance protein identification requires the use of usually gel-free mass spectrometry (MS)-based approaches. Using Arabidopsis thaliana as a model system, in this chapter, we describe all the steps followed for the extraction of microsomal proteins to MS analysis of separated peptides with a major focus on three different methods, namely, OFFGEL fractionation, 2D-LC, and long-column method for the identification of low-abundance proteins. Separation of such peptides will lead to in-depth proteomics-based investigations to answer biological questions.