Chemical gradients play an important role in nature, driving many.different phenomena critical to life, including the transport of Chendeal species across membranes and the transport, attachment, and assembly of cells. Taking a cue from these natural processes, scientists and engineers are now working to develop synthetic chemical gradients for use in a broad range of applications, such as in highthroughput investigations of surface properties, as means to guide the motions and/ or assembly of liquid droplets, vesicles, nanoparticles, and cells and as new media for stationaryphasegradient chemical separations. Our groups have been working to develop new methods for preparing chemical gradients from organoalkoxysilane and organochlorosilane precursors and to obtain a better understanding of their properties on macroscopic to microscopic length scales. This review highlights our recent work on the development of controlledrate infusion and infusionwithdrawal dipcoating methods for the preparation of gradients on planar glass and silicon substrates, on thinlayer chromatography plates, and in capillaries and monoliths for liquid chromatography. We also cover the new knowledge gained from the characterization of our gradients using sessile drop and Wilhelmy plate dynamic water contact angle measurements, Xray photoelectron spectroscopy mapping, and singlemolecule tracking and spectroscopy. Our studies reveal important evidence of phase separation and cooperative interactions occurring along multicomponent gradients. Emerging concepts and new directions in the preparation and characterization of organosilane-based chemical gradients are also discussed.