Fibrillar collagens and glycosaminoglycans (GAGs) are structural biomolecules that are natively abundant to the extracellular matrix (ECM). Prior studies have quantified the effects of GAGs on the bulk mechanical properties of the ECM. However, there remains a lack of experimental studies on how GAGs alter other biophysical properties of the ECM, including ones that operate at the length scales of individual cells such as mass transport efficiency and matrix microstructure. Here we characterized and decoupled the effects of the GAG molecules chondroitin sulfate (CS) dermatan sulfate (DS) and hyaluronic acid (HA) on the stiffness (indentation modulus), transport (hydraulic permeability), and matrix microarchitecture (pore size and fiber radius) properties of collagen-based hydrogels. We complement these biophysical measurements of collagen hydrogels with turbidity assays to profile collagen aggregate formation. Here we show that CS, DS, and HA differentially regulate the biophysical properties of hydrogels due to their alterations to the kinetics of collagen self-assembly. In addition to providing information on how GAGs play significant roles in defining key physical properties of the ECM, this work shows new ways in which stiffness measurements, microscopy, microfluidics, and turbidity kinetics can be used complementary to reveal details of collagen self-assembly and structure.