Nitroxyl (HNO) has received significant recent attention due to its remarkable and novel biological activity and pharmacological potential. Unlike most other commonly studied nitrogen oxides, examination of HNO biology is not straightforward because of its unique chemical properties. Thus, the study of HNO in biological system requires researchers to be aware of this chemistry in order to properly design and interpret experiments. This chapter focuses on the experimental issues associated with the study of HNO and attempts to provide guidelines for working with this novel and fascinating nitrogen oxide.