Active-learning strategies such as undergraduate research courses and course-based undergraduate research experiences (CUREs), which engage students in the practical experiments, have been reported as efficient ways for effective teaching and training of graduating students. Recently, many practical examples have been published in various fields to develop critical skills needed by undergraduate students for graduate school or the workforce; however, very few examples have been published for bioanalytical topics, specifically, Liquid Chromatography-Mass Spectrometry (LC-MS-MS). Herein, we describe an innovative undergraduate research course that was used to train well-prepared graduating seniors on LC-MS-MS and in developing a sensitive method for detecting vitamin B9 (folic acid) levels in different milk samples. During this semester-long exercise, the students were exposed to literature review, basic UV-Vis spectroscopy, and HPLC and LC-MS-MS techniques. Additionally, as part of this laboratory-based research course, they researched published approaches on analyzing folic acid in food samples and devised a method to extract and quantify folic acid levels in different brands of milk using LC-MS-MS instruments. Feedback from students and faculty members was very positive as the students felt that this opportunity helped them to become more comfortable using HPLC and LC-MS-MS instruments and felt empowered to answer real-life analytical problems. This laboratory-based undergraduate research course can also be modified and used as an advanced bioanalytical laboratory exercise in biochemistry or analytical chemistry courses.