To date, few general chemistry laboratory studies have included affective measures despite calls for more research on aspects of this domain. This shortage of studies may be partially due to the scarcity of affective measures that have been designed for, or tested in, the college laboratory setting. To provide measures for use in this environment, several existing affective scales were adapted for this new context. Before data from the scales were utilized to study the environment, evidence was provided for the validity and reliability of the data generated from them. Once sufficient evidence was provided, it was possible to determine affective differences between students completing a Beer’s law experiment in the traditional hands-on laboratory (control group) and a similar experiment in a virtual environment (treatment group). To assess expected differences between environments, scales for anxiety, emotional satisfaction, intellectual accessibility, usefulness of lab, equipment usability, and open-endedness of lab were selected. To account for potential between-student differences, scales for feeling-related initial interest and value-related initial interest were selected. Overall, students who completed the virtual experiment scored significantly lower on the emotional satisfaction, intellectual accessibility, usefulness of lab, and equipment usability scales. However, it was noted that student responses in the virtual environment varied significantly by which teaching assistant (TA) instructed the section. To test for a possible instructor effect, data from the virtual sections were grouped by TA as “Virtual Group A” and “Virtual Group B”. Group A contained the TAs who had sections with lower averages on the emotional satisfaction scale as compared to group B. After controlling for instructor, differences between student responses in the “Hands-On” sections and “Virtual Group A” sections were no longer significant while significant differences remained between the responses in the “Hands-On” and “Virtual Group B” sections. This outcome indicated that the TA instructing the course may have been more influential on students’ affective outcomes than the environment in which the experiment was performed.