Pediatric cancers are a group of diseases of genetic origin that represent the first cause of death in the pediatric population in developed countries. Particularly, acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood. Correct diagnosis of ALL is of vital importance for the success of the treatment. It relies on the identification of factors associated with the risk of disease relapse after remission has been achieved and with the treatment response, and the further use of those factors to adapt to the intensity of treatment. Nowadays, large experimental analyses of proteins such as mass spectrometry and proteomics techniques permit a more precise identification of proteins associated with a disease. This work represents a useful review about analysis of protein samples from pediatric patients diagnosed with acute lymphoblastic leukemia (ALL), through mass spectrometry-based proteomic methodologies.