There are several villages in Spain called Guadalupe, but I suppose that you mean the best known of them, located in the province of Caceres, Extremadura region, a village with a Parador and a Monastery.
The best way to get there from Madrid is by bus. The company that serves the route is
La Sepulvedana . It runs twice a day (8am and 3pm) and the journey takes 3:45 hours. On Fridays, there is one more departure at 7pm. One-way fare, about 14 euro.
The buses depart from Estacion Sur de Autobuses (Metro station: Mendez Alvaro).