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Introduction: Workload can be defined as the hypothetical construct that represents the cost incurred by a human operator to achieve a particular level of performance. Several methods have been used to assess the workload. One of the most common methods of assessing mental workload is NASA-TLX method. The present study was conducted to develop and validate of a pilot activity load index (PALI) based on NASA-TLX template. Materials and Methods: This study was a cross-sectional and descriptive-analytical that was carried out among airline pilots of Civil Aviation Organization. At first, “Pilot Activity Load Index” or PALI subscales were developed based on NASA-TLX template. Validity of the subscales was evaluated by Content Validity Ratio (CVR) and Content Validity Index (CVI).Using Lawshe’s table. Finally, to determine the internal consistency of the subscales, 30 pilots completed the final version of the PALI in typical flight phases. The data obtained were then analyzed by SPSS using Cronbach's alpha to measure the reliability of the subscales and considering an acceptance level of 0.7. Results: The first results led to the emergence of an initial list with 17 subscales. Eleven subscales were irrelevant, redundant and impractical to use in operational environment, due to CVR less than 0.75 and were omitted from the subscales list. Finally, six PALI subscales were approved related to pilot tasks (CVI=0.79). The Verified subscales had a Cronbach's alpha value of 0.89 and were therefore considered a reliable tool. Conclusion:  The results of this study showed that PALI questionnaire which validity and reliability has been approved can be used for assessment of workload in pilots.