Various analytical tools have been leveraged in single cell analysis to study cell-to-cell variations. Among the different types of cells, sperm makes an attractive candidate for single cell analysis owing to its high heterogeneity. It is known that while about 100 million sperm are found in a single ejaculate, generally only one sperm fertilizes the oocyte and develops into embryo. Additionally, no two sperm can yield genetically identical embryo. The fact that only one sperm is involved in the fertilization process and that sperms within the same ejaculate are highly heterogeneous suggests that single sperm analysis essential. However, despite the growing interest, research and clinical practice in male fertility has mostly relied on population, or sample-based characteristics. While different methods have been developed to perform single cell analysis, isolating and analyzing many individual single sperm still remains a challenge due to the constant motion of the cells. In this presentation, I will present a microfluidic trap array that is capable of capturing and aligning a large number of live sperms in an organized and referenceable format. The method enables high-resolution imaging of the aligned cell head, DNA and mitochondrial analyses via fluorescence staining, and the quantification of tail beating characteristics. In testing, an array of 2400 trapped ~ 400 sperm for individual analyses of tail beating frequency and amplitude, DNA integrity via acridine orange (AO) staining, and mitochondrial activity via Mito-Tracker Deep Red staining. The microfluidic trap array demonstrated here uniquely enables the high-throughput study of individual live sperm in semen samples.