Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are ubiquitous in eukaryotes, and their assembly in a four-helix bundle drives membrane fusion. To monitor in real-time the folding of these proteins, we have developed a new versatile self-contained SNAREpin and have performed optical tweezers experiments on four representative sets of SNAREs: (i) Synaptic, (ii) GLUT4, (iii) early endosome, and (iv) yeast. (i) In neurons, SNAREs are tightly regulated to achieve fusion of the synaptic vesicles within one millisecond upon arrival of the action potential. (ii) GLUT4 is a highly effective glucose transporter responsible for most of glucose clearance in blood in response to insulin. (iii) Early endosome SNAREs are key players in membrane recycling between cell organelles. (iv) Yeast SNARE is believed to be representative of the proto-SNAREs that have expanded in metozoa during adaption to multicellular lifestyle. Despite the fact that biological context of the different sets of SNAREs is very different in respect to their regulation, as well as their time constrains for fusion and energetic costs, we have found that the structural and physical properties of their assembly is highly conserved. In all SNAREs investigated, assembly proceeds from the N to C terminal of the bundle and is punctuated by three sequential binary switches: the amino- and carboxy-terminal followed by a linker domain. Finally, the energetic and structural pathways of the assembly we have quantified provide details on how SNAREs drives fusion and are regulated within the SNARE superfamily.