In patients with acute lymphoblastic leukemia (ALL), treatment response is increasingly evaluated with minimal residual disease (MRD) assays. ALL cells can be recognized by their clonal rearrangement of immunoglobulin and T-cell receptor genes, expression of gene fusions, and leukemia-associated immunophenotypes. Assays based on polymerase chain reaction or flow cytometry can detect one ALL cell among 10,000 to 100,000 normal cells in clinical samples. The vast majority of cases have antigen-receptor gene rearrangements and leukemia immunophenotypes for MRD monitoring; about half of the cases currently have suitable gene fusions. The clinical significance of MRD has been conclusively demonstrated in both childhood and adult ALL. In most studies, MRD positivity is defined by the presence of 0.01% or more ALL cells; the risk of relapse is generally proportional to the level of MRD, particularly when measured during or at the end of remission-induction therapy. The prevalence of MRD during early therapy differs among genetic and biologic ALL subtypes. However, being a measurement of drug resistance in vivo and reflecting multiple cellular, host, and treatment variables, MRD is typically an independent prognostic factor. MRD is now used in several clinical trials for risk assignment and to guide clinical management overall. The time points at which MRD testing is performed and the threshold levels that trigger treatment intensification vary according to the methodology available, the results of preclinical correlative studies, and protocol design.