Liposomal anticancer agents can effectively deliver drugs to tumor lesions, but their therapeutic effects are enhanced in only limited number of patients. Appropriate biomarkers to identify responder patients to these liposomal agents will improve their treatment efficacies. We carried out pharmacological and histopathological analyses of mouse xenograft models bearing human ovarian cancers (Caov‐3, SK‐OV‐3, KURAMOCHI, and TOV‐112D) to correlate the therapeutic effects of doxorubicin‐encapsulated liposome (Doxil®) and histological characteristics linked to the enhanced permeability and retention effect. We next generated 111In‐encapsulated liposomes to examine their capacities to determine indications for Doxil® treatment by single‐photon emission computed tomography (SPECT)/CT imaging. Antitumor activities of Doxil® were drastically enhanced in Caov‐3, moderately in SK‐OV‐3, and minimally in KURAMOCHI and TOV‐112D when compared to doxorubicin. Microvessel density and vascular perfusion were high in Caov‐3 and SK‐OV‐3, indicating a close relation with the enhanced antitumor effects. Next, 111In‐encapsulated liposomes were given i.v. to the animals. Their tumor accumulation and area under the curve values over 72 h were high in Caov‐3, relatively high in SK‐OV‐3, and low in two other tumors. Importantly, as both Doxil® effects and liposomal accumulation varied in the SK‐OV‐3 group, we individually obtained SPECT/CT images of SK‐OV‐3‐bearing mouse (n = 11) before Doxil® treatment. Clear correlation between liposomal tumor accumulation and effects of Doxil® was confirmed (R2 = 0.73). Taken together, our experiments definitely verified that enhanced therapeutic effects through liposomal formulations of anticancer agents depend on tumor accumulation of liposomes. Tumor accumulation of the radiolabeled liposomes evaluated by SPECT/CT imaging is applicable to appropriately determine indications for liposomal antitumor agents. Advantages of liposomal anti‐cancer agents include increased anti‐tumor activities through EPR effect, and reduced side effects associated with their free drugs; however, despite these clear benefits, we are unequivocally reminded that liposomal agents enhance therapeutic effects to the limited number of cancer patients. Here, we performed pharmacological and histopathological analyses of mouse xenograft models bearing human ovarian cancers to correlate therapeutic effects of Doxil and histological characteristics linked to EPR, and we subsequently generated radiolabeled liposomes to examine their capacities as biomarkers to determine indications for Doxil treatment by SPECT/CT imaging. Our preclinical study clearly indicates the correlations among pharmacological effects of Doxil, histological factors linked to EPR, tumor accumulation of the radiolabeled liposomes, as well as SPECT/CT tests of the radiolabeled liposomes, and demonstrates applicability of SPECT/CT imaging to properly decide the indications for liposomal anti‐cancer agents with significant potential towards the clinical practice.