Endotoxin content is a critical factor that affects the safety of biological pharmaceutical products. International pharmacopoeias describe several reference methods to determine endotoxin levels in advanced therapy medicinal product (ATMP) preparations. Administration of ATMPs must be done as rapidly as possible to ensure complete viability and potency of the cellular product. To evaluate the endotoxin content in the shortest time possible, we chose to validate an alternative method based on the use of the Charles River Portable Testing System (PTS) and FDA-approved cartridges, compliant with the requirements of the European Pharmacopoeia and providing results in <20 min. Here, we describe a unique and complete validation approach for instrument, personnel, and analytical method for assessment of endotoxins in ATMP matrices. The PTS system provides high sensitivity and fast quantitative results and uses less raw material and accessories compared with compendial methods. It is also less time consuming and less prone to operator variability. Our validation approach is suitable for a validated laboratory with trained personnel capable of conducting the ATMP release tests, and with very low intra-laboratory variability, and meets the criteria required for an alternative approach to endotoxin detection for in-process and product-release testing of ATMPs.