The purpose of this study was to develop a scoring rubric to assess students' manipulation skills and identify students' learning difficulties in conducting organic chemistry experiments. In constructing the scoring rubric, we first analyzed the skills needed in the experiment, then divided the skills into subskills, and finally constructed a rubric for each subskill. We applied this developed instrument to on-site evaluation of 55 second-year students by eight teaching assistants (TAs). The data were analyzed using one-way analysis of variance, which compared the students' mean score on each skill at the 0.05 significance level. Results showed that the skill of gravity filtration and suction filtration needed to be improved, as the students had the lowest mean scores in these two skills. On-site observations further indicated that only 5% of the students correctly performed the rinsing technique for gravity filtration. We also found that TAs became more aware of students' experimental skills after using the rubric for assessment. By using a scoring rubric as an assessment tool, students' learning difficulties could be easily identified and TAs could improve their teaching effectiveness by checking student performance and modifying their instructional strategies accordingly. This study provides a valuable and easy-to-apply quantitative assessment for use in the organic chemistry laboratory.