4-hydroxynonenal (4-HNE) and 6-hydroxydopamine (6-OHDA)-mediated damage in dopaminergic neurons is well documented. Protective potential of steroidal hormone (17β-estradiol) has also been suggested. However, therapeutic potential of such promising hormone is hampered due to complex brain anatomy and physiology. Thus, the present investigations were studied to suggest the applicability of dopamine expressing PC12 cells as in vitro tool to screen the pharmacological potential of 17β-estradiol against 4-HNE and 6-OHDA. MTT assay was conducted for cytotoxicity assessment of both 4-HNE (1 μM to 50 μM) and 6-OHDA (10(-4) to 10(-7) M). Non-cytotoxic concentrations, that is, 4-HNE (1 μM) and 6-OHDA (10(-6) M) were selected to study the synergetic/additive responses. PC12 cells were found to be more vulnerable towards co-exposure of individual exposure of 4-HNE and 6-OHDA, even at non-cytotoxic concentrations. Then, cells were subjected to pre-treatment (24 hours) of 17β-estradiol (1 μM), followed by a permutation of combinations of both 4-HNE and 6-OHDA. Pretreatment of 17β-estradiol was found to be significantly effective against the cytotoxic responses of 4-HNE and 6-OHDA, when the damage was at lower level. However, 17β-estradiol was found to be ineffective against higher concentrations. Physiological-specific responses of PC12 cells against 4-HNE/6-OHDA and 17β-estradiol suggest its applicability as first tier of screening tool.