Childhood acute lymphoblastic and myeloid leukemias are stratified into molecular and cytogenetic subgroups important for prognosis and therapy. Studies have shown that gene expression profiles can discriminate between leukemia subtypes. Thus, proteome analysis similarly holds the potential for characterizing different subtypes of childhood leukemia. We used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to analyze cell lysates from childhood leukemia cell lines as well as pretreatment leukemic bone marrow derived from childhood leukemia cases. Comparison of the acute myeloid leukemia (AML) cell line, Kasumi, and the biphenotypic myelomonocytic cell line, MV4;11, with the acute lymphoblastic leukemia (ALL) cell lines, 697 and REH, revealed many differentially expressed proteins. In particular, one 8.3 kDa protein has been identified as a C-terminal truncated ubiquitin. Analysis of childhood leukemia bone marrow showed differentially expressed proteins between AML and ALL, including a similar peak at 8.3 kDa, as well as several proteins that differentiate between the ALL t(12;21) and hyperdiploid subtypes. These results demonstrate the potential for proteome analysis to distinguish between various forms of childhood leukemia. Future analyses are warranted to validate these findings and to investigate the role of the C-terminal truncated ubiquitin in the etiology of ALL.