Background and Purpose: The Nurse Practitioner Holistic Caring Instrument (NPHCI) is a 19-item, investigator-developed instrument designed to measure holistic caring in nurse practitioner (NP) practice. This paper evaluates multi-sample psychometric testing of the instrument, describing data from three samples, with analysis supporting the NPHCI as a valid and reliable instrument. Methods: The NPHCI has been administered in patient, NP program faculty, and NP convenience samples. Development of the NPHCI was guided by Shuler’s NP practice model and Swanson’s theory of caring. Construct validity was estimated using Interitem correlation, exploratory factor analysis and confirmatory factor analysis. Reliability of the instrument was assessed in all three samples via Cronbach’s alpha. Findings: Exploratory factor analysis consistently supported the identification of three subscales in the NPHCI, and confirmatory factor analysis demonstrated a good fit between the theoretical constructs and empirical indicators in the data. The instrument has demonstrated strong reliability in three samples. Conclusions: The NPHCI demonstrates acceptable reliability and validity but further psychometric testing is indicated.