The knowledge of the load in prestressed bolted connections is essential for the proper operation and safety of engineering structures. Recently, bolted joints have become an area of intensive research associated with non-destructive diagnostics, in particular in the context of wave propagation techniques. In this paper, a novel procedure of bolt load estimation based on the energy of Lamb wave signals was proposed. Experimental tests were performed on a single lap joint of two steel plates. Ultrasonic waves were excited and registered by means of piezoelectric transducers, while precise measurement of the bolt load was obtained by means of using the force washer transducer. Experimental tests were supported by the finite element method analysis based on Schoenberg’s concept. The results showed that the relationship between the bolt load and signal energy was strongly nonlinear and it depended on the location of acquisition points.