Many methods have been proposed to extract and quantify algal pigments. Comparative studies have found that pigment extraction efficiency varies among solvent and mechanical disruption protocols due to differential cellular resistance, thereby, leading to potential misinterpretation of pigment data. When the type or resistance of algae are unknown, a method is required that efficiently extract pigments from all taxonomic groups. The objective of this study was to develop a simple and efficient one stage periphyton pigment extraction protocol by comparing the extractability of four solvents (acetone, methanol, methanol/acetone, and methanol/acetone/N,N-dimethylformamide), the effects of grinding, and the effects of freeze-drying. The best overall extraction was obtained using freeze-dried samples extracted with methanol/acetone/DMF/water (MAD). Eighty-six percent more chlorophyll was extracted when the sample was freeze-dried relative to fresh/frozen samples extracted with 90% acetone. Freeze-drying greatly improved the extraction of both polar and non-polar (lipophilic/hydrophobic) pigments while MAD increased the extractability of polar pigments and improved peak resolution of all pigments. Chemotaxonomic assessment differed between samples that were fresh/frozen or freeze-dried before extraction. The relative abundance of cyanobacteria was greater for freeze-dried material compared with fresh/frozen due to the improved extractability of cyanobacterial pigments. Based on the results of this study, the traditional approach of 90% acetone as a solvent is not recommended for periphyton samples containing cyanobacteria or when the composition of the mat is unknown. The combination of freeze-drying and MAD was sufficient for the extraction of pigments from a periphyton mat containing filamentous cyanobacteria, green algae, and diatoms.