The folate inhibitor methotrexate (MTX) is an important component of osteosarcoma (OS) treatment regimens. New generation multitargeted antifolates, such as pemetrexed (PMX), have shown promise in the treatment of various solid tumors. In this study, the in vitro efficacy of MTX and PMX was compared in OS cell lines. MTX demonstrated a superior cytotoxic effect in comparison to PMX in all tested cell lines. Apoptosis assays revealed that both MTX and PMX induce apoptosis but MTX demonstrated superior efficacy. These in vitro results suggest that PMX as a single agent may not demonstrate improved efficacy compared to MTX in OS patients.