The lack of a viable theory for describing diffusivity when fluids are confined at the micro- and nanoscale [Ladero et al. Chem. Eng. Sci.2007, 62, 666-678; Deen AIChE J.1987, 33, 1409-1425] has necessitated accurate measurement of diffusivity (D) [Jin and Chen Chromatographia2000, 52, 17-21; Nie et al. Science1994, 266, 1018-1021; Durand et al. Anal. Chem.2009, 81, 5407-5412], crucial for a host of micro- and nanofluidic technologies [Grattoni et al. Curr. Pharm. Biotechnol.2010, 11, 343-365]. We demonstrate a rapid and agile method for the direct measurement of diffusivity in a system possessing 104 to 105 precisely fabricated channels with characteristic sizes (β) ranging from micro- to nanometers. Custom chambers allowed us to measure the diffusivity in a closed unperturbed system using UV/vis spectroscopy. D was measured for rhodamine B (RhoB) in aqueous solution in channels of 200 and 1 μm, as well as 13 and 5.7 nm. The observed logarithmic scaling of diffusivity with β, in close agreement with prior experiments, but far from theoretical prediction, surprisingly highlights that diffusivity is significantly altered even at the microscale. Accurate measurement of D by reducing the size of the source reservoir by 3 orders of magnitude (from 150 μL to 910 nL) proves that a substantial reduction in measurement time (from 7 days to 40 min) can be achieved. Our design thus is ready for rapid translation into a standard analytical tool - useful for multiple applications.