Understanding the knowledge, attitudes, and practices (KAP) of hospital staff is needed to improve care activities that support the detection/prevention/treatment of malnutrition, yet quality measures are lacking. The purpose was to develop (study 1) and assess the administration and discriminative potential (study 2) of using such a KAP measure in acute care. In study 1, a 27-question KAP questionnaire was developed, face validated (n = 5), and tested for reliability (n = 35). Kappa and Intraclass Correlation (ICC) were determined. In study 2, the questionnaire was sent to staff at five diverse hospitals (n = 189). Administration challenges were noted and analyses completed to determine differences across sites, professions, and years of practice. Study 1 results demonstrate that the knowledge/attitude (KA) and the practice (P) subscales are reliable (KA: ICC = 0.69 95% CI 0.45–0.84, F = 5.54, p < 0.0001; P: ICC = 0.84 95% CI 0.68−0.92, F = 11.12, p < 0.0001). Completion rate of individual questions in study 2 was high and suggestions to improve administration were identified. The KAP mean score was 93.6/128 (range 51–124) with higher scores indicating more knowledge, better attitudes and positive practices. Profession and years of practice were associated with KAP scores. The KAP questionnaire is a valid and reliable measure that can be used in needs assessments to inform improvements to nutrition care in hospital.