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Absorption spectroscopy is one of the most widely used techniques employed for determining the concentrations of absorbing species (chromophores) in solutions. It is a nondestructive technique which biologists and biochemists and now systems biologists use to quantify the cellular components and characteristic parameters of functional molecules. This quantification is most relevant in the context of systems biology. For creating a quantitative depiction of a metabolic pathway, a number of parameters and variables are important and these need to be determined experimentally. This chapter describes the UV–visible absorption spectroscopy used to produce experimental data for bottom-up modeling approaches of systems biology which uses concentrations and kinetic parameters (K_m and V_max) of enzymes of metabolic/signaling pathways, intracellular concentrations of metabolites and fluxes. It also briefly …