Summary Background Despite the expansion of capnography use, instruments for evaluating healthcare professionals’ knowledge about capnography are missing from international literature. Objectives To develop and validate an instrument for evaluating nurses’ knowledge about capnography, named the “Nurses’ Knowledge about Capnography Test” (NKCT). Method A literature review was performed to formulate instrument items. Thirty-six items were initially developed, which covered principles of capnography function, conditions affecting end-tidal CO2 pressure and capnography waveform, and indications for capnography use. Six items were deleted after content validity and intra-rater reliability evaluation. The final 30-item instrument was completed by 103 anesthesiology department nurses employed in six hospitals of Greece, to test its internal consistency, item difficulty and discrimination, and construct validity. Results Kuder–Richardson 20 coefficient was 0.79. Ten items were found to be of high difficulty, while item discrimination was low for two of them. Instrument scores were found significantly higher among participants with higher educational level and longer experience in capnography use. Conclusion NKCT is a psychometrically comprehensive instrument for evaluating nurses’ knowledge about capnography, which is recommended to be re-validated beyond the anesthesiology department and be used for the assessment of educational programs on capnography.