Background: Although recombinant tissue plasminogen activator has become the first-line thrombolytic agent, streptokinase remains in use in acute ST-elevation myocardial infarction (STEMI) patients in Malaysian public hospitals. In a state hospital in Malaysia, all post-STEMI patients given streptokinase are counselled and issued with a streptokinase card by pharmacists. Due to its highly antigenic nature, patient knowledge of streptokinase and the streptokinase card serve as important alerts to healthcare providers. This study aimed to develop a validated questionnaire on evaluating knowledge of streptokinase and the streptokinase card among post-STEMI patients. Methods: A Malay language questionnaire was developed based on literature review and a standard streptokinase counselling checklist. The questionnaire consisted of three parts: demographics, knowledge of streptokinase and understanding of streptokinase card. The content of the questionnaire was revised by eight experts (emergency specialists, physicians and pharmacists) and six patients were interviewed via telephone, 2 weeks after being issued with a streptokinase card, to test for comprehensibility. Then, a telephone survey was conducted during June-December 2017 involving another 60 patients to test for construct validity and reliability. Results: The questionnaire was finalised by decreasing the overall number of items from 6 to 5 (Section B) and 6 to 4 (Section C). The overall measurement of sample adequacy (MSA) was 0.758. Bartlett's test was highly significant (<0.001). The Cronbach's alpha values for Sections B and C were 0.747 and 0.841, respectively. Conclusion: Our nine-item questionnaire is a valid and reliable instrument to assess knowledge of streptokinase and the streptokinase card among Malaysian post-STEMI patients.