||One difficulty that many laboratories face with expanded newborn screening is a high number of false positive test results. Modifying cutoffs has been used to reduce the false positive rate, but this is done at the expense of increasing the number of false negative test results. In this research, second-tier tests-defined as alternative methods used to test the same blood spot as the primary screening with no additional patient contact-were developed for homocystinruia and for maple syrup urine disease. The test for homocystinuria is specific for total homocysteine, and the test for maple syrup urine disease is specific for hydroxyproline, allo-isoleucine, isoleucine, and leucine. Parameters, such as sensitivity, specificity, precision, accuracy, reportable range, carryover, reference interval and ion suppression were analyzed for each second-tier test. Results indicate that both of these tests are acceptable for clinical testing, and that these tests should be helpful in differentiating true positives from false positives. It is thus recommended that these second-tier tests for homocystinuria and maple syrup urine disease be implemented in the clinical setting.