Müller cells, the principal glia of the retina, generate tractional forces in response to IGF-I and platelet-derived growth factor and are present in diabetic fibro-vascular scar tissues causing traction retinal detachment. While diabetes-associated increases in vitreous IGFs have been reported, paradoxically high concentrations of these same growth factors in normal vitreous suggest the presence of more complex mechanisms regulating growth factor bioavailability. To define diabetes-associated changes in vitreous biological activity, the stimulatory effects of 68 samples were evaluated using Müller cell tractional force generation as a target bioassay. Dose-response profiles were used to calculate vitreous specific activity (per unit protein) and total vitreous activity (per unit volume). Vitreous samples from patients lacking diabetes or other retinal pathology had undetectable or low activities, whereas diabetic retinopathy was associated with 6.9- and 8.7-fold increases in vitreous specific and total activities, respectively. Secondary analyses revealed no activity differences associated with patient sex, age, or the presence of vitreous hemorrhage. However, compared with diabetes alone, the presence of proliferative diabetic retinopathy was associated with additional 2.3-fold increases in vitreous specific and total activities. Vitreous dose-response assays performed with and without growth factor-neutralizing antibodies enable attribution of vitreous activity to IGFs (53.9%) and, to a lesser extent, platelet-derived growth factors (14.5%). Because the observed increases in vitreous growth factor activity grossly exceed the reported increases in growth factor concentration, these data indicate that diabetes-associated changes in vitreous biological activity involve more complex biochemical changes that ultimately yield increased growth factor bioavailability and/or Müller cell responsiveness.