Quantitative and reliable measurement of cellular invasion is important to understand a range of biological processes such as cancer metastasis and angiogenesis. Spheroid invasion assays are an attractive in vitro platform because they effectively mimic the tumor cell invasion of solid tissues. Here, we developed an image analysis-based method to quantify the invasiveness of HT1080 human fibrosarcoma tumor cell spheroids. We segmented a cell-covered area into three subareas using objectively set threshold pixel intensities and calculated invasion indices using these subareas. Comparison with conventional parameters for spheroid invasion assays, such as area, length, and detached cells, showed that our indices present the invasion event at an early time and without being convoluted by proliferation. As an application, we then examined paracrine interactions between LLC1 mouse lung carcinoma cells and Raw264.7 mouse macrophage cells with our developed analysis method. We found that the invasion of tumor spheroids was increased by a macrophage-conditioned medium, concomitantly with a decrease in tumor cell proliferation. Importantly, invasion was further enhanced by a conditioned medium from activated macrophages by co-culture with tumor cells. Thus, our indices reveal that tumor cell invasion is facilitated in a feed-forward manner by communication between tumor cells and macrophages in the tumor microenvironment.