Cybersickness is a drawback of virtual reality (VR), which also affects the cognitive and motor skills of users. The Simulator Sickness Questionnaire (SSQ) and its variant, the Virtual Reality Sickness Questionnaire (VRSQ), are two tools that measure cybersickness. However, both tools suffer from important limitations which raise concerns about their suitability. Two versions of the Cybersickness in VR Questionnaire (CSQ-VR), a paper-and-pencil and a 3D–VR version, were developed. The validation of the CSQ-VR and a comparison against the SSQ and the VRSQ were performed. Thirty-nine participants were exposed to three rides with linear and angular accelerations in VR. Assessments of cognitive and psychomotor skills were performed at baseline and after each ride. The validity of both versions of the CSQ-VR was confirmed. Notably, CSQ-VR demonstrated substantially better internal consistency than both SSQ and VRSQ. Additionally, CSQ-VR scores had significantly better psychometric properties in detecting a temporary decline in performance due to cybersickness. Pupil size was a significant predictor of cybersickness intensity. In conclusion, the CSQ-VR is a valid assessment of cybersickness with superior psychometric properties to SSQ and VRSQ. The CSQ-VR enables the assessment of cybersickness during VR exposure, and it benefits from examining pupil size, a biomarker of cybersickness.