<em>Capsicum annuum</em> is a crop species of economic importance able to produce capsaicinoids, capsinoids, and pigments with nutritional and medicinal value. Methods to propagate and transform this species have been reported, but most are phenotype dependent, rely on <em>Agrobacterium</em> for transformation, and their success has been limited. This relates to only one commercial transgenic variety currently on trial. In the present work, we report the conditions to produce callus and cell suspension cultures of <em>C. annuum</em> ‘Serrano’ using commercial seeds. The culture could be induced to produce capsaicin and dihydrocapsaicin in detectable quantities and was amenable to transformation using biolistics. The expression of the <em>Arabidopsis thaliana</em> soluble inorganic pyrophosphatase 4 fused to a fluorescent protein was demonstrated using confocal microscopy. Evidence of the integrity of the fusion was obtained by immunoblot. The transformation induced a change in the ratio of capsaicin to dihydrocapsaicin measured using high resolution direct sample analysis-mass spectrometry (DSA-MS). The method is thus useful for the study of capsaicinoid production under controlled conditions for special purposes and metabolic studies.