Great improvements have been made in acute lymphoblastic leukemia (ALL) treatment in the past decades, especially due to the use of L-asparaginase (L-ASP). Despite the significant success rate, several side effects mainly caused by toxicity, asparaginase silent inactivation, and cellular resistance, encourage an open debate regarding the optimal dosage and formulation of L-ASP. Alternative sources of asparaginases have been constantly investigated in order to overcome hypersensitivity clinical toxicity. Additionally, genomic modulation as gene expression profiling, genetic polymorphisms, and epigenetic changes is also being investigated concerning their role in cellular resistance to L-ASP. Understanding the mechanisms that mediate the resistance to L-ASP treatment may bring new insights into ALL pathobiology and contribute to the development of more effective treatment strategies. In summary, this review presents an overview on L-ASP data and focuses on cellular mechanisms underlying resistance and alternative therapies for the use of asparaginase in childhood ALL treatment.