We present a detailed investigation of the performance of lens-free holographic microscopy toward high-throughput on-chip blood analysis. Using a spatially incoherent source that is emanating from a large aperture, automated counting of red blood cells with minimal sample preparation steps at densities reaching up to ~0.4 × 10 6 cells/μL is presented. Using the same lens-free holographic microscopy platform, we also characterize the volume of the red blood cells at the single-cell level through recovery of the optical phase information of each cell. We further demonstrate the measurement of the hemoglobin concentration of whole blood samples as well as automated counting of white blood cells, also yielding spatial resolution at the subcellular level sufficient to differentiate granulocytes, monocytes, and lymphocytes from each other. These results uncover the prospects of lens-free holographic on-chip imaging to provide a useful tool for global health problems, especially by facilitating whole blood analysis in resource-poor environments.Together with several recent contributions, lens-free imaging 1-11 is now becoming more and more important, especially for cytometry and microscopy applications. Among others, one field that would enormously benefit from lens-free on-chip digital imagers is the field of microfluidics. 12,13 Over the past decade, microfluidics has revolutionized the available toolset to handle cells by significantly reducing the required device and reagent volumes, as well as the associated costs. Lens-free on-chip imaging could provide further reduction in the size and the cost of these microfluidic systems, while, at the same time, improving the throughput of imaging and/or characterization. To complement these advances in lens-free imaging and microfluidics, recently, we have introduced an on-chip holographic microscopy platform with subcellular resolution over a large field of view (~24 mm 2 ). [8][9][10][11] In this technique, by controlling the spatial coherence of the illumination source (which can be a simple lightemitting diode), we record the two-dimensional (2D) holographic diffraction signature of each cell or microparticle on an opto-electronic sensor array without the use of any lenses or lasers (see Figure 1). This recorded holographic pattern is then rapidly processed using a customdeveloped algorithm 9,10 to perform wide field-of-view (FOV) microscopy of semitransparent objects such as blood cells on the chip.Providing a spatial resolution of ~1.4-1.5 μm, 9,11 which is comparable to a 10× objective-lens with a numerical aperture of ~0.1-0.2 at ~500-600 nm, but with an order-of-magnitude-larger * Author to whom correspondence should be addressed. FOV (e.g., 24 mm 2 vs ~2 mm 2 ), our lens-free microscopy platform is capable of imaging a large number of cells simultaneously. As a result, this system meets the requirements and needs of whole blood analysis applications, which inevitably necessitate high-throughput imaging and characterization. Toward this end, here, we presen...