Heparanase-1 (HPSE-1), an endo-β-d-glucuronidase, is the only known enzyme that cleaves heparan sulfate (HS) [1,2]. The HPSE-1 gene encodes a preproenzyme, that is converted to a proenzyme (ProHPSE-1) in the endoplasmic reticulum. The proenzyme is then shuttled to the Golgi apparatus and secreted outside of the cell to bind to HS at the cellular surface. After internalization [3,4] and transportation to endosomes then fused with lysosomes [5], ProHPSE-1 is activated by Cathepsin L to give the enzymatically active mature HPSE-1 [6]. Active HPSE-1 is located primarily in lysosomes or late endosomes and can also be found in the nucleus [7] and autophagosomes [8]. In response to local or systemic cues, active HPSE-1 can be secreted outside of the cell, where it plays a pivotal role in the degradation and remodeling of the extracellular matrix (ECM) and basement membranes (BM). Additionally, via cleavage of HS chains, HPSE-1 aids in the release of active biological molecules that are sequestered by HS chains in the cellular microenvironment [9]. Under normal conditions, HPSE-1 is strictly regulated, and is involved in physiological functions including wound-healing and tissue remodeling [10], embryonic morphogenesis [11], leukocyte trafficking [12, 13, 14, 15], and hair growth [16]. Pathologically, HPSE-1 activity plays a significant role in inflammatory diseases [17] and the progression of cancer and metastasis [18]. Consequently, HPSE-1 has become very attractive as a therapeutic target, and growing evidence suggests its inhibition has therapeutic value for various pathological conditions [19]. Therefore, methods to interrogate HPSE-1 activity are important to propel drug discovery efforts of HPSE-1 inhibitors and further understanding of the functions of HPSE-1. A number of methods have been developed to measure HPSE-1 activity in vitro using different types of HPSE-1 substrates [20]. Herein, we will summarize these chemical tools in three categories depending on the nature of the HPSE-1 substrate: (I) polysaccharide-based, (II) fondaparinux (FPX)-based, and (III) disaccharide-based, while highlighting the HPSE-1 probes recently developed in our lab.