High‐density lipoproteins (HDLs) isolated by immunoaffinity chromatography and separated by immobilized pH gradient‐isoelectric focusing (IPG‐IEF) were examined by mass spectrometry directly, applying a new proteomics technology, virtual two‐dimensional (2‐D) gel electrophoresis. A preliminary examination of HDL particles has revealed at least 42 unique masses for protein species with isoelectric points between pH 5.47–5.04, some of which have not been observed previously. By delivering masses of intact proteins from complex cellular mixtures in a format that correlates directly to classical 2‐D gel analyses, virtual 2‐D gel electrophoresis constitutes a general discovery tool to expose and monitor protein isoforms and post‐translational modifications. Furthermore, its general ability to deliver ions from sub‐picomole level proteins enmeshed in complex cellular mixtures potentially fulfills the need of top‐down proteomics to obtain intact protein ions from microscale samples. Additional comparison of such data to 2‐D gel analyses and their identified proteins may elucidate the functions of the individual apolipoprotein components and the cardioprotective effects of HDL.