Developing, homeostatic, and regenerating tissues are full of various gradients, including mechanical, chemical, porosity and growth-factor gradients. However, it remains challenging to replicate these gradients using common tissue engineering approaches. Here, we use electrospinning to create scaffolds with in-depth gradients. We created a fiber diameter gradient and pore size gradient throughout the depth of electrospun (ESP) scaffolds by a continuous gradient of polymer concentration. As an alternative to this established method, we developed a novel method to create fiber diameter gradients by changing the voltage on both needle and collector, keeping the total voltage constant. In this way, fiber diameter could be changed in a gradient matter by focusing the electrospinning spot. Using this method, we created a fiber diameter and pore size gradient, while keeping all other parameters constant. Lastly, we developed a novel method to create functional group gradients, which can potentially be used in a wide variety of polymer solutions to couple peptides and proteins to ESP scaffolds. A scaffold with an in-depth gradient of functional groups was created by adding functionalized poly(ethylene glycol) additives to the polymer solution, a novel method with potentially wide applications. The techniques demonstrated here could be applied to a wide variety of polymers and applications and can aid in developing physiologically relevant gradient scaffolds.