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(1) Objective: Parental alienation phenomenon was given an increased attention during the recent years in both the clinical and the legal fields. This study aims to introduce the development of a new parental alienation questionnaire. (2) Method: Four studies were performed. In the first two studies in which five specialists participated, the questionnaire was defined, and the inter-rater reliability was verified. The next study, which included 267 participants, sought to establish the construct validity by applying Exploratory Factor Analysis and Confirmatory Factor Analysis. The last study that enrolled 200 participants considered predictive validity as well as test-retest reliability. (3) Results: Eight criteria of parental alienation have been identified: Campaign of denigration, frivolous, weak, or absurd rationale for the alienation, lack of ambivalence towards the alienating, lack of guilt or remorse about the alienation, borrowed scenarios, independent thinker phenomenon, taking the alienating parent’s side in the conflict, and spread of alienation to the extended family of the targeted parent. (4) Conclusion: The results highlighted the proper psychometric qualities of the questionnaire. The Parental Alienation Questionnaire (PAQ) seems to be a promising tool not only for clinical and judicial practice, but also for research.