The present study developed and evaluated the content validity of a survey questionnaire titled, “Instrument to Assess Teachers’ Practice of Differentiated Instruction (IATPDI).” The items of IATPDI questionnaire were adapted from the pre-existing survey questionnaires and existing literatures on differentiated instruction. To quantify content validity of IATPDI questionnaire, a pre-developed survey questionnaire with 40 items was emailed to two established content experts to: (1) rate each item in terms of its clarity and relevancy to the measured domain; (2) evaluate which item should be deleted or revised; and (3) get recommendation about whether additional items are needed to adequately tap the domain of interest. The Delphi method was used to collect the data. Subsequently, two rounds of Delphi process culminated in a revised and refined IATPDI questionnaire with 40 simplified items divided into four sections. Content validity of IATPDI questionnaire was quantified by calculating content validity index (I-CVI and S-CVI) and modified kappa statistics, which indicated high content validity of the 40 items. Taken together, the results show that the IATPDI questionnaire is a content valid instrument.