Soluble guanylyl cyclase (sGC, also called GC1) is the main receptor for nitric oxide (NO) that catalyzes the production of the second messenger molecule, 3′5′ cyclic guanosine monophosphate (cGMP) leading to vasorelaxation, and inhibition of leukocyte recruitment and platelet aggregation. Enhancing cGMP levels, through sGC agonism or inhibition of cGMP breakdown via phosphodiesterase inhibition, has yielded FDA approval for several cGMP modifier therapies for treatment of cardiovascular and pulmonary diseases. While basic research continues to improve our understanding of cGMP signaling and as new therapies evolve to elevate cGMP levels, we provide a short methodological primer for measuring cGMP and cGMP-mediated vascular relaxation for investigators. • Nitric oxide (•NO) stimulates soluble guanylyl cyclase (sGC) to catalyze the production of cyclic guanosine 3′, 5′-monophosphate (cGMP) • We provide general guidelines for assessing sGC-cGMP signaling pathway in cells and tissue as well as measuring ex vivo •NO-sGC-cGMP mediated vascular relaxation • By combining cell and tissue-based measurements of cGMP and myography, one can rigorously assess the impact of new physiological factors and pharmacological modulators that influence •NO -cGMP signaling [ABSTRACT FROM AUTHOR]