BACKGROUND Flow cytometry essentially focuses on surface-expressed proteins, with few protocols being devoted to intracellular components. We evaluated a two-step procedure using new formaldehyde-free permeabilization and staining reagents that allow the staining of platelets and red blood cells (RBCs) from whole blood. METHODS Citrated blood was treated with the new staining protocol (NSP) or control reagent (phosphate-buffered solution bovine serum albumin) and stained with antibodies against surface or intracellular markers. The effects of the NSP on cell integrity, morphology, and content were evaluated. RESULTS The NSP slightly reduced the cell count (similar to 20%) and changed the RBC morphology with a 42% mean diameter reduction. Conversely, the NSP did not affect platelet discoid morphology and led to a minor size decrease (11%). These morphological changes neither impelled a gating strategy modification nor interfered with the discrimination among populations based on surface markers. The NSP provided intracellular access to all the tested antigens: CD62P, FXIII, and CD63 in platelets and glycated and fetal hemoglobin (HbA1c and HbF) and nucleic acid in RBCs. The NSP gave excellent intra-assay precision with minimal impact on cell morphology and fluorescence labelling over time (up to 24 h). CONCLUSIONS With the ability to detect surface and intracellular antigens through a rapid preparation protocol without washing steps or toxic formaldehyde treatment, this NSP designed for research offers a marked improvement in the analysis of platelets and RBCs isolated directly from whole blood. Consequently, the NSP opens new avenues to investigate platelet degranulation and erythrocyte subpopulations.