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Objective: Approximately 80% of patients with pancreatic adenocarcinoma (PA) are diagnosed with locally advanced, unresectable, or metastatic disease at presentation. First-, second-, and third-line chemotherapies are beneficial for patients. Gemcitabine (GEM)-based regimens played an indispensable role over the last two decades. There are several options for second-line treatment, such as a combination of oxaliplatin, fluorouracil (FU), and folinic acid (FOLFOX). However, the prognosis is still poor. Therefore, GEM as a targeted reference should be a reasonable potential approach. This study aimed to establish a first-line, second-line treatment model for future drug screening. Materials and Methods: We cultured SW1990 cells and compared the inhibitory effect on cell proliferation between the GEM-FOLFOX group, i.e., GEM treatment was followed by FOLFOX addition, and FOLFOX-GEM group, i.e., GEM was added after FOLFOX. Furthermore, we evaluated the inhibitory effect of both groups by changing onset of treatment with drugs, i.e., at early start (24 h after seeding cells) with that at delayed start (48 h after seeding cells), or by changing treatment duration i.e., long treatment duration (24 h) with that of shortened treatment duration (12 h). We quantified the expression of an antiapoptotic gene B cell CLL/lymphoma 2 (Bcl-2) using Real-Time quantitative polymerase chain reaction (PCR). Results: The GEM-FOLFOX group showed higher inhibition than the FOLFOX-GEM group, irrespective of delayed treatment or shortened treatment duration. Bcl-2 expression was more inhibited in the GEM-FOLFOX group. Conclusions: We developed a first-line, second-line treatment model for screening novel drugs that could have similar or better futuristic potential than GEM.