Background: Chronic Kidney Disease is a complex condition characterized by a decrease in kidney function. Objective: The objective of this study was to develop and validate a knowledge tool and assess the knowledge of pharmacists regarding Chronic Kidney Disease (CKD). Methods: The study consisted of 2 phases with phase-I involving the development and validation of a knowledge tool. The development phase encompassed a thorough review of the literature, focus-group discussion, and expert review. The validation phase consisted of pilot testing and involving content and face validity and reliability testing using Cronbach alpha. Phase-II involved an assessment of knowledge of pharmacists by administering the validated tool to 183 practicing pharmacists working in tertiary care hospitals in 3 big cities of Pakistan. The data obtained were statistically analyzed involving descriptive and inferential statistics including chi-square test, one way ANOVA, post hoc Tukey’s test, and regression analysis. Results: The developed knowledge was found to be reliable with Cronbach alpha values above 0.7 during the pilot testing. A high response rate of 93.4% was observed during the study. 50.9% of the pharmacists in the study had good knowledge, the mean percent knowledge score was found to be 54.76. The knowledge score was found to be associated with the male gender, 30 – 39 years of age, >5 years of experience and clinical / hospital pharmacy certification during the post hoc analysis (p<005). Strong positive correlation and linear regression relationship were observed between percentage knowledge score and number of years of professional clinical experience. Conclusions: Pharmacists working in tertiary care hospitals in Pakistan exhibit a good knowledge of chronic kidney disease.