In response to concerns regarding effects of hyperlinked annotation on reading comprehension, this study was undertaken to compare hyperlinked annotation with student highlighting of unknown/difficult words. An online highlighting tool was used to help students reflect their prior vocabulary in a hyperlink-based annotated passage. Highlighting unknown/difficult words was expected to aid acquisition of those words, increase the reading comprehension of the passages, and decrease overall cognitive load. Japanese undergraduates (n = 80) were randomly assigned to each of following four conditions: treatment groups with and without a pretest vocabulary quiz, and control groups with and without the pretest. A Solomon four-group design was used because the pretest vocabulary quiz might provide students with time to reflect their prior knowledge and could, therefore, reduce the effect of the treatment. Results showed that highlighting unknown/difficult words in a hyperlink-based annotated passage was not effective on its own for enhancing vocabulary learning. On the other hand, the combination of a pretest vocabulary quiz and highlighting unknown/difficult words increased reading comprehension, and decreased perceived cognitive load. The vocabulary quiz was not intended to be part of the treatment but was discovered to be important as an adjunct to highlighting unknown/difficult words.