The research combines systematization of existing publications and the results obtained earlier by the authors to propose a new approach to assessment of alienation and behavioral opportunism of personnel at modern enterprises and corporations. The paper presents a rationale for assessing the four components of social alienation (self-estrangement, powerlessness, social isolation, and meaninglessness), and six forms of behavioral opportunism: four for opportunism by commission (lying or misrepresentation, violation of agreements, exaggeration of difficulties, using unforeseen events for benefits), as well as two forms of opportunism by omission (information hiding and negligence). The authors propose to use a combination of surveys with direct (to evaluate the degree of alienation, sixteen questions, four for each component) and indirect (to evaluate willingness for behavioral opportunism, six questions with five answer options each) questions and a quantitative assessment of the degree of alienation and the level of behavioral opportunism. We regarded opportunism as a complex of two phenomena (the subjects’ willingness to opportunism and the conditions for its manifestation). We considered structural elements of both phenomena and proposed tools for their measurement, also substantiating their interrelation. We propose approaches to the assessment and interpretation of the results obtained during the application of the proposed methodology. A feature of the proposed methodology is the extensive possibilities of its use in practice, including for monitoring the corporate culture of modern enterprises. It allows assessing the degree of alienation and the level of behavioral opportunism and social exclusion both for an individual worker and for their groups (departments, representatives of one profile, separately for employees and managers), as well as for the enterprise as a whole. Of particular importance is the proposed approach for assessing the corporate culture and the quality of institutions existing in a corporation at the level of interaction of its main subjects: employees, managers, and owners.