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Colorectal cancer (CRC) is a disease with constantly increasing incidence and highmortality. The treatment efficacy could be curtailed by drug resistance resulting from poordrug penetration into tumor tissue and the tumor-specific microenvironment, such ashypoxia and acidosis. Furthermore, CRC tumors can be exposed to different pHdepending on the position in the intestinal tract. CRC tumors often share upregulationof the Akt signaling pathway. In this study, we investigated the role of external pH in controlof cytotoxicity of perifosine, the Akt signaling pathway inhibitor, to CRC cells using 2D and3D tumor models. In 3D settings, we employed an innovative strategy for simultaneousdetection of spatial drug distribution and biological markers of proliferation/apoptosisusing a combination of mass spectrometry imaging and immunohistochemistry. In 3Dconditions, low and heterogeneous penetration of perifosine into the inner parts of thespheroids was observed. The depth of penetration depended on the treatment durationbut not on the external pH. However, pH alteration in the tumor microenvironment affectedthe distribution of proliferation- and apoptosis-specific markers in the perifosine-treatedspheroid. Accurate co-registration of perifosine distribution and biological response in thesame spheroid section revealed dynamic changes in apoptotic and proliferative markersoccurring not only in the perifosine-exposed cells, but also in the perifosine-free regions.Cytotoxicity of perifosine to both 2D and 3D cultures decreased in an acidic environmentbelow pH 6.7. External pH affects cytotoxicity of the other Akt inhibitor, MK-2206, in asimilar way. Our innovative approach for accurate determination of drug efficiency in 3Dtumor tissue revealed that cytotoxicity of Akt inhibitors to CRC cells is strongly dependenton pH of the tumor microenvironment. Therefore, the effect of pH should be consideredduring the design and pre-clinical/clinical testing of the Akt-targeted cancer therapy.