Background: Item analysis is the set of qualitative and quantitative techniques and procedures used to evaluate the characteristics of items of the test before and after the test development and construction. Multiple Choice Questions (MCQs) are a popular assessment tool due to their high objectivity, high reliability and the ability to assess a large content in a short time span. Aims and objectives: The aim of the present study was to to validate the MCQs/Item . Material and methods: 120 MCQs in Biochemistry of the single best response type were subjected to the process of item analysis and the difficulty index, discrimination index and distracter effectiveness were calculated using standard formulae. These MCQs and distracters were then classified into groups as per standard reference ranges of these parameters and the absolute number and percentage of MCQs and distracters in each group were calculated. Results: The percentage of MCQs that could be accepted as having desired validity based on difficulty index, discrimination index were 78(65%), 24(20%). Thus these MCQs could be used for assessment while the rest needed modification and retesting or needed to be discarded. Conclusion: MCQ item analysis must be performed as it helps in question paper setting as per the need of assessment, it improves Teaching ? Learning outcomes and is a source of great item bank.