Heparin and low-molecular weight heparins (LMWHs), complex, sulfated polysaccharides isolated from endogenous sources, are potent modulators of hemostasis. Heparin and LMWHs interact with multiple components of the coagulation cascade to inhibit the clotting process. Pharmaceutical preparations of these complex polysaccharides, typically isolated from porcine intestinal mucosa, are heterogeneous in length and composition and, hence, highly polydisperse. Because of the structural heterogeneity of heparin and LMWHs, correlating their activity with a particular structure or structural motif has been a challenging task. Herein, we demonstrate a practical analytical method that enables the measurement of a structural correlate to in vivo anticoagulant function. With this understanding we have developed LMWHs with increased anticoagulant activity and decreased polydispersity. In addition to the pronounced anti-Xa and anti-IIa activity of these LMWHs, we also demonstrate that they possess desirable in vivo pharmacokinetic properties, the ability to cause the release of tissue factor pathway inhibitor (TFPI) from the endothelium, complete bioavailability through s.c. delivery, and the ability to inhibit both venous and arterial thromboses. Importantly, from a clinical safety point of view, unlike LMWHs presently used in the clinic, we show that these LMWHs are rapidly and completely neutralized by protamine. Together, the findings presented herein demonstrate a facile approach for the creation of designer LMWHs with optimal activity profiles.