Our first aim was to present a simple and quantitative data analysis method with a new potential in the application of liver SPECT imaging. We have established quantitative SPECT/CT in vivo imaging protocols for determination of liver tumor burden based on the known role of Kupffer cells in cancer of the liver. As it is also known that functional Kupffer cells accumulate particulate material contained in the arterial blood of liver supply, we used 99mTc radiolabeled macro-aggregated albumin (MAA) particles intravenously. An other aim was to find statistical parameters based on activity maps that could estimate the present of cancerous cells either in semi-quantitative imaging. We try to show that these parameters show the presence of tumorous cells before it is visible in SPECT/CT image as a „nodular” lesion. We examined a healthy control group and group of induced hepatocellular carcinoma (HCC), where hepatocellular carcinoma was induced by diethylnitrosamine and a group with melanoma xenograft . We used 99mTc-MAA as radiopharmaceutical for liver SPECT imaging and eXIA160 contrast material for liver CT imaging using a small animal SPECT/CT system. Segmentation of the liver was calculated by Otsu thresholding method. Based on the segmentation the radioactivity volume, summarized liver activity, the skewness and the kurtosis of the activity distribution were determined. Ex vivo liver mass data were applied for the validation of in vivo measurements. An uptake with cold spots as tumors was observed in all HCC animals in SPECT/CT scans. Cold spots are not visible in the HM group. Isotope labeled particle uptake (standardized uptake concentration – SUC), the skewness and the kurtosis of control and tumorous groups was significantly different. Conclusion: a new potential application of 99mTc-MAA was developed and presents a simple and very effective means to characterize liver cold spot lesions and show the possibility of cancerous cells resulting from Kupffer cell pathological distribution as a consequence of tumor burden.