Quantitative analysis and modeling of signaling pathway components can reveal important information about the dynamics of that system, including the relative stoichiometries and affinities between the individual signaling components, as well as rate-limiting steps in the signaling pathway. In this chapter, we present a method that we developed to quantify the steady-state ratio of core Hedgehog (Hh) signaling components in both cultured cells and the Drosophila embryo, a physiologically relevant tissue.