Peri-prosthetic joint infection (PJI) definition plays an important role in diagnostic and therapeutic decisions. However, while several criteria have been proposed by eminent institutions to define a PJI in the last decade, their clinical validation has been rarely performed. Aim of the present multicenter, international, retrospective study was to validate the World Association against Infection in Orthopedics and Trauma (WAIOT) pre/intra-operative PJI definition with post-operative confirmatory tests. A total of 210 patients, undergoing hip ( = 86) or knee ( = 124) revision surgery for any reason in six orthopedic centers in Africa, Asia, Europe and South America, were retrospectively evaluated at a two years minimum follow-up after surgery. All the available pre-, intra- and post-operative findings were collected and analyzed according to the WAIOT criteria, which include a set of tests to confirm (Rule In) or to exclude (Rule Out) a PJI. On average, patients were investigated pre/intra-operatively with 3.1 ± 1.1 rule out and 2.7 ± 0.9 rule in tests; the presence of a fistula or exposed implant was reported in 37 patients (17.6%). According to pre/intraoperative findings, 36.2% of the patients were defined as affected by high-grade PJI ( = 76; average score: 2.3 ± 0.8), 21.9% by low-grade PJI ( = 46; average score: 0.8 ± 0.8), 10.5% by biofilm-related implant malfunction ( = 22; average score: -1.6 ± 0.8), 2.9% as contamination ( = 6; average score: -3.5 ± 1.0), and 28.6% as no infection ( = 60; average score: -3.0 ± 1.4). Pre/intra-operative PJI definitions matched post-operative confirmatory tests, in 97.1% of the patients. This is, to our knowledge, one of the largest study ever conducted to validate a PJI definition The retrospective analysis in different centers was greatly facilitated by the structure of the WAIOT definition, that allows to include different tests on the basis of their sensitivity/specificity, while the comparison between pre/intra-operative and post-operative findings offered the internal validation of the scoring system. Our results authenticate the WAIOT definition as a reliable, simple tool to identify patients affected by PJI prior to joint revision surgery.