Researchers have shown that current instruments used to assess an individual’s level of differentiation of self or differentiation are biased towards an individualistic cultural context, therefore current instruments lack cultural validity (Alaedein, 2008; Chung & Gale, 2006, 2009; Kim et al., 2015; O’Hara & Meteyard, 2011). When counseling from Bowen’s family systems theoretical perspective, accurate assessment of an individual’s differentiation is integral for treatment planning and intervention (Bowen & Kerr, 1988). To address the existing gap in assessment of differentiation within multiple cultural contexts (i.e., individualistic, collectivistic, transcultural), the purpose of the present research was to develop the Multicultural Differentiation of Self Inventory (MDSI) and conduct initial validation. Graduate students, 22 years and older from 33 universities in southeast United States (U.S.), completed a Demographic Questionnaire (i.e., university, age, gender, socio-economic status, relationship status, race, ethnicity, country of birth, residence state in U.S., residence country/countries, current geographic location due to Covid-19 pandemic, country/countries of citizenship, language(s) spoken, immigrant generation in the U.S. from another country, cultural affiliation, current majority community cultural setting), the 36-item Multicultural Differentiation of Self Inventory (MDSI), and the 16-item Individualism-Collectivism Revised Scale (INDCOL-R; Triandis & Gelfand, 1998). Results of an exploratory factor analysis using a principle components analysis indicated that the MDSI was not a valid instrument to assess differentiation of self in the individualistic, collectivistic, and transcultural contexts. Further, validity and reliability of the INDCOL-R Scale was established. Cronbach’s alpha indicated good reliability (i.e., .70 or higher) for all four dimensions, including horizontal individualism (M = 26.78, SD = 5.92), horizontal collectivism (M = 28.21, SD = 5.09), vertical individualism (M = 17.97, SD = 6.65), and vertical collectivism (M = 23.69, SD = 6.65), as well as individualism (M = 44.75, SD = 9.85) and collectivism (M = 51.88, SD = 9.31). Using Pearson’s r and the probability cutoff value of .01 and .001, INDCOL-R Scale and dimension correlations ranged from very weak (.12, p = .01; vertical individualism and vertical collectivism) to very strong (.85, p = .001; collectivism and vertical collectivism). Also using Pearson’s r, INDCOL-R Scale and demographics correlations ranged from very weak (.12, p = .01; vertical collectivism and socio-economic status; INDCOL-R Scale and current majority community cultural setting; respectively) to weak (.21, p = .001; vertical collectivism and relationship status). Future research is needed to develop a culturally valid differentiation of self instrument