Abstract: Novel insights into complex biological processes very often critically depend on the establishment of new potent read-out tools and improved protocols. A lot has been learned over the past four decades on physiological functions and, importantly, disease-related roles of the prion protein (PrP), a relatively broadly expressed membrane-anchored glycoprotein with high levels in several cell types of the nervous and immune system and with well-established key roles in different progressive and fatal neurodegenerative protein misfolding diseases (proteopathies). However, while several controversies and unclarities remain even for these widely accepted involvements, currently unexplored (and unexpected) facets and functions may still wait to be discovered. New light might be shed into these aspects by a better understanding of potential intrinsic roles of previously largely unconsidered post-translationally generated forms or fragments of PrP, for instance those resulting from endogenous proteolytic cleavage (Mohammadi et al., 2022; Vanni et al., 2022). In fact, membranebound full-length (FL) PrP, the form most research of the past has focused on, may not even represent the majority of total PrP in the brain (Vanni et al., 2022). A nearly FL form released from cells by a constitutive and very membrane-proximate proteolytic cleavage event (“shedding”) makes up for a rather small yet relevant fraction and is of emerging interest. This physiological, anchorless, and predominantly double-glycosylated form, now called “shed PrP” (sPrP), has repeatedly been reported in the past (e.g., Parizek et al., 2001), for instance, in cell culture media supernatants or body fluids, yet its mechanistic origin and biological relevance remained obscure for a long time. The latter, to a great deal, is due to technical challenges differentiating this fragment from excess FL-PrP present in most biological specimens (e.g., tissue homogenates; but even in body fluids or cell culture supernatants, FLPrP is present on cellular membrane debris and physiologically released extracellular vesicles (EVs)). Both forms are of similar molecular weight and share structure and sequence and, hence, epitopes for most available antibodies used for detection in standard laboratory techniques (Mohammadi et al., 2022). Besides recently improved protocols to differentiate and quantify the abundance of different PrP “proteoforms” by immunoblotting (Vanni et al., 2022), cleavage site-directed antibodies previously presented for the reliable detection of rodent sPrP have become a convenient tool to systematically, highly specifically, and comparably comfortably assess sPrP with various methods, as these antibodies are “blind” for the just few amino acids (plus the C-terminally attached glycosylphosphatidylinositol anchor) longer FL form (Linsenmeier et al., 2021; Mohammadi et al., 2022). However, given that many key aspects regarding PrP shedding, such as cleavage site and responsible protease, remained uncharacterized for the human body, our groups recently set out to unravel many unknowns in this regard. We eventually succeeded in identifying the shedding site (Y226↓Q227) in human PrP and recently presented an in-depth characterization of respective antibodies exclusively detecting sPrP (Song et al., 2024). As expected from previous mouse data, we revealed that the shedding of human PrP is likewise strictly dependent on the metalloprotease ADAM10, and we did not find any evidence for alternative proteolytic cleavages in the vicinity of Y226 (Song et al., 2024).