Diphenidine [1-(1,2-diphenyl-ethyl)piperidine] in the tissue sections of the adipose, brain, heart, kidney, and liver obtained from a human cadaver in a poisoning case was directly analyzed as the first trial by matrix-assisted laser desorption ionization (MALDI)-quadrupole time-of-flight mass spectrometry (QTOF-MS). The frozen tissue block of approximately 5 × 5 × 5 mm3 was sectioned to a thickness of 10 or 20 μm at −20 or −35 °C. For quantitation, the reference standard solutions of diphenidine were placed at 0, 0.1, 1, and 10 ng/well on the wells of a MALDI sample plate firstly, and about 20 tissue sections (ca. 5 × 5 mm2 each) were placed on the sample plate secondly, followed by detection by MALDI-MS/MS after desiccation of the tissue sections and spraying α-cyano-4-hydroxycinnamic acid as the matrix. The quantitation was performed by the standard addition method without the use of blank human tissues. The correlation coefficients between the concentrations of diphenidine and the relative signal intensities were from 0.937 to 0.994 for five kinds of tissue. The suppression of signals by tissue was increased in the order of adipose tissue < brain < heart, kidney, and liver; hence, the adipose tissue was most suitable tissue among them for the detection of diphenidine. In the present method, the concentration linearity and repeatability were not satisfactory for quantitative analysis, but allowable for semiquantitation. Therefore, the present method can be used for a semiquantitative analysis of diphenidine in tissue sections because of the simplicity of the method.