Identifying students' conceptual scientific understanding is difficult if the appropriate tools are not available for educators. Concept inventories have become a popular tool to assess student understanding; however, traditionally, they are multiple choice tests. International science education standard documents advocate that assessments should be reform based, contain diverse question types, and should align with instructional approaches. To date, no instrument of this type targeting student conceptions in biotechnology has been developed. We report here the development, testing, and validation of a 35-item Biotechnology Instrument for Knowledge Elicitation (BIKE) that includes a mix of question types. The BIKE was designed to elicit student thinking and a variety of conceptual understandings, as opposed to testing closed-ended responses. The design phase contained nine steps including a literature search for content, student interviews, a pilot test, as well as expert review. Data from 175 students over two semesters, including 16 student interviews and six expert reviewers (professors from six different institutions), were used to validate the instrument. Cronbach's alpha on the pre/posttest was 0.664 and 0.668, respectively, indicating the BIKE has internal consistency. Cohen's kappa for inter-rater reliability among the 6,525 total items was 0.684 indicating substantial agreement among scorers. Item analysis demonstrated that the items were challenging, there was discrimination among the individual items, and there was alignment with research-based design principles for construct validity. This study provides a reliable and valid conceptual understanding instrument in the understudied area of biotechnology.