Osteoarthritis (OA) is a chronic, progressive disease which has over 300 million cases each year. Some of the main symptoms of OA are pain, restriction of joint motion and stiffness of the joint. Early diagnosis and treatment can prolong painless joint function. Vibroarthrography (VAG) is a cheap, reproducible, non-invasive and easy-to-use tool which can be implemented in the diagnostic route. The aim of this study was to establish diagnostic accuracy and to identify the most accurate signal processing method for the detection of OA in knee joints. In this study, we have enrolled a total of 67 patients, 34 in a study group and 33 in a control group. All patients in the study group were referred for surgical treatment due to intraarticular lesions, and the control group consisted of healthy individuals without knee symptoms. Cartilage status was assessed during surgery according to the International Cartilage Repair Society (ICRS) and vibroarthrography was performed one day prior to surgery in the study group. Vibroarthrography was performed in an open and closed kinematic chain for the involved knees in the study and control group. Signals were acquired by two sensors placed on the medial and lateral joint line. Using the neighbourhood component analysis (NCA) algorithm, the selection of optimal signal measures was performed. Classification using artificial neural networks was performed for three variants: I—open kinetic chain, II—closed kinetic chain, and III—open and closed kinetic chain. Vibroarthrography showed high diagnostic accuracy in determining healthy cartilage from cartilage lesions, and the number of repetitions during examination can be reduced only to closed kinematic chain.